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Sample records for mass labeled 13c14-decabromodiphenylethane

  1. Mass Spectrometry-Based Label-Free Quantitative Proteomics

    PubMed Central

    Zhu, Wenhong; Smith, Jeffrey W.; Huang, Chun-Ming

    2010-01-01

    In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies. PMID:19911078

  2. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  3. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

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

    PubMed Central

    Basu, Sankha S; Blair, Ian A

    2013-01-01

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

  5. Effect of tumor mass and antigenic nature on the biodistribution of labeled monoclonal antibodies in mice

    SciTech Connect

    Watanabe, Y.; Endo, K.; Koizumi, M.; Kawamura, Y.; Saga, T.; Sakahara, H.; Kuroki, M.; Matsuoka, Y.; Konishi, J.

    1989-06-01

    The effect of tumor mass and antigenic nature on the biodistribution of 111In- and 125I-labeled monoclonal antibodies (MoAbs) was studied using F(ab')2 fragments of three representative anti-tumor MoAbs and SW1116 human colorectal carcinoma grown in nude mice. The 19-9, F33-104 anti-CEA, and 17-1A MoAbs showed specific binding to SW1116 cells. The former two MoAbs recognize circulating CA 19-9 with molecular weights of more than 5,000,000 and CEA of Mr 170,000-180,000, respectively, whereas 17-1A reacts with a nonshedding antigen. Both percentage injected dose per gram tumor and tumor-to-blood ratios were inversely proportional to the tumor mass in nude mice administered 111In- and 125I-labeled 19-9, but liver uptake increased as tumor size increased. Analysis of serum samples and tumor homogenates demonstrated the presence of a high-molecular-weight species, probably due to the antibody binding to CA 19-9. In the case of 111In-labeled anti-CEA MoAb, tumor uptake also decreased and liver uptake increased with tumor size, but this effect was less obvious than that of 19-9. In contrast, tumor and liver uptake of 125I-labeled anti-CEA MoAb, 111In- and 125I-labeled 17-1A and control antibodies were independent of tumor mass. The absolute tumor uptake and tumor-to-blood ratios of all 125I-labeled antibodies were lower than those of the 111In-labeled ones. And the effect of tumor mass was also weaker with 125I-labeled antibodies, probably due to in vivo dehalogenation. These results indicate that the effect of tumor size on the incorporation of labeled MoAb into tumors is dependent on the antigenic nature to be targeted and/or radionuclides used for labeling and that high concentrations of circulating high molecular weight antigens may limit in vivo use of MoAb conjugates.

  6. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

    PubMed Central

    Zhou, Yuping; Vachet, Richard W.

    2012-01-01

    Covalent labeling and mass spectrometry are seeing increased used together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g. diethylpyrocarbonate) and non-specific (e.g. hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues, and thus protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g. 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g. microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. As compared to typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 Å to 7 Å for myoglobin, 13 Å to 10 Å for

  7. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

    NASA Astrophysics Data System (ADS)

    Zhou, Yuping; Vachet, Richard W.

    2012-04-01

    Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for

  8. Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics.

    PubMed

    Robinette, David; Neamati, Nouri; Tomer, Kenneth B; Borchers, Christoph H

    2006-08-01

    Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design. PMID:16901199

  9. INVESTIGATION OF ARSINE-GENERATING REACTIONS USING DEUTERIUM-LABELED REAGENTS AND MASS SPECTROMETRY

    EPA Science Inventory

    Mass spectrometry was used to detect transfer of deuterium from labeled reagents to arsines following hydride-generation reactions. The arsine gases liberated from the reactions of arsenite, arsenate, methylarsonic acid, and dimethylarsinic acid with HC1 and NaBD4 in H2O, or with...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Exploring membrane protein structural features by oxidative labeling and mass spectrometry.

    PubMed

    Konermann, Lars; Pan, Yan

    2012-10-01

    Despite their biological importance, the structural characterization of integral membrane proteins (IMPs) by x-ray crystallography and NMR spectroscopy remains challenging. Hence, there is a need for complementary approaches that are capable of probing IMP conformational features in a robust fashion. Covalent labeling relies on the principle that solvent accessible regions can be modified by reactive species, whereas buried segments are protected. The readout of the labeling pattern is conducted by mass spectrometry. Hydroxyl radical (·OH) introduces oxidative modifications at amino acid side chains. In this article, the authors discuss the application of ·OH labeling for the structural interrogation of IMPs. Kyte-Doolittle hydropathy analyses are widely used for generating IMP topology models. The validation of these models by mutational techniques is labor intensive. ·OH labeling can readily distinguish transmembrane elements from solvent-exposed loops, thereby providing an alternative topology validation tool. For IMPs with published crystal structures, oxidative modifications can report on functionally relevant dynamic features that are invisible in the static x-ray data. The coupling of pulsed ·OH labeling with rapid mixing techniques represents a novel approach for studying IMP folding kinetics. In conclusion, ·OH labeling is a versatile tool that can provide insights into the structure and dynamics of IMPs. PMID:23194267

  12. Label-free imaging, detection, and mass measurement of single viruses by surface plasmon resonance

    PubMed Central

    Wang, Shaopeng; Shan, Xiaonan; Patel, Urmez; Huang, Xinping; Lu, Jin; Li, Jinghong; Tao, Nongjian

    2010-01-01

    We report on label-free imaging, detection, and mass/size measurement of single viral particles in solution by high-resolution surface plasmon resonance microscopy. Diffraction of propagating plasmon waves along a metal surface by the viral particles creates images of the individual particles, which allow us to detect the binding of the viral particles to surfaces functionalized with and without antibodies. We show that the intensity of the particle image is related to the mass of the particle, from which we determine the mass and mass distribution of influenza viral particles with a mass detection limit of approximately 1 ag (or 0.2 fg/mm2). This work demonstrates a multiplexed method to measure the masses of individual viral particles and to study the binding activity of the viral particles. PMID:20798340

  13. Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry.

    PubMed

    Wanigasekara, Maheshika S K; Chowdhury, Saiful M

    2016-09-01

    Arginine residues undergo several kinds of post-translational modifications (PTMs). These PTMs are associated with several inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, and diabetes. Mass spectrometric studies of arginine modified proteins and peptides are very important, not only to identify the reactive arginine residues but also to understand the tandem mass spectrometry behavior of these peptides for assigning the sequences unambiguously. Herein, we utilize tandem mass spectrometry to report the performance of two widely used arginine labeling reagents, 1,2-cyclohexanedione (CHD) and phenylglyoxal (PG) with several arginine containing peptides and proteins. Time course labeling studies were performed to demonstrate the selectivity of the reagents in proteins or protein digests. Structural studies on the proteins were also explored to better understand the reaction sites and position of arginine residues. We found CHD showed better labeling efficiencies compared to phenylglyoxal. Reactive arginine profiling on a purified albumin protein clearly pointed out the cellular glycation modification site for this protein with high confidence. We believe these detailed mass-spectrometric studies will provide significant input to profile reactive arginine residues in large-scale studies; therefore, targeted proteomics can be performed to the short listed reactive sites for cellular arginine modifications. PMID:27543028

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2015-07-01

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

  16. freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis

    PubMed Central

    Li, Zhenye; Pan, Chao; Duan, Huilong

    2015-01-01

    Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range. PMID:26665161

  17. Interconversion of Peptide Mass Spectral Libraries Derivatized with iTRAQ or TMT Labels.

    PubMed

    Zhang, Zheng; Yang, Xiaoyu; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Ji, Weihua; Markey, Sanford P; Roth, Jeri; Neta, Pedatsur; Hizal, Deniz Baycin; Bowen, Michael A; Stein, Stephen E

    2016-09-01

    Derivitization of peptides with isobaric tags such as iTRAQ and TMT is widely employed in proteomics due to their compatibility with multiplex quantitative measurements. We recently made publicly available a large peptide library derived from iTRAQ 4-plex labeled spectra. This resource has not been used for identifying peptides labeled with related tags with different masses, because values for virtually all masses of precursor and most product ions would differ for ions containing the different tags as well as containing different tag-specific peaks. We describe a method for interconverting spectra from iTRAQ 4-plex to TMT (6- and 10-plex) and to iTRAQ 8-plex. We interconvert spectra by appropriately mass shifting sequence ions and discarding derivative-specific peaks. After this "cleaning" of search spectra, we demonstrate that the converted libraries perform well in terms of peptide spectral matches. This is demonstrated by comparing results using sequence database searches as well as by comparing search effectiveness using original and converted libraries. At 1% FDR TMT labeled query spectra match 97% as many spectra against a converted iTRAQ library as compared to an original TMT library. Overall this interconversion strategy provides a practical way to extend results from one derivatization method to others that share related chemistry and do not significantly alter fragmentation profiles. PMID:27386737

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

    PubMed

    Huan, Tao; Li, Liang

    2015-07-21

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

  19. Validation of membrane protein topology models by oxidative labeling and mass spectrometry.

    PubMed

    Pan, Yan; Ruan, Xiang; Valvano, Miguel A; Konermann, Lars

    2012-05-01

    Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms. PMID:22410873

  20. Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Pan, Yan; Ruan, Xiang; Valvano, Miguel A.; Konermann, Lars

    2012-05-01

    Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ṡOH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ṡOH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

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

    PubMed

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

    2016-01-28

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

  2. Comprehensive discovery of 13C labeled metabolites in the bacterium Methylobacterium extorquens AM1 using gas chromatography-mass spectrometry.

    PubMed

    Yang, Song; Hoggard, Jamin C; Lidstrom, Mary E; Synovec, Robert E

    2013-11-22

    Herein, we report the identification of isotopically labeled metabolite peaks (or the lack of labeling) between sets of GC-MS data from Methylobacterium extorquens AM1. M. extorquens AM1 is one of the best-characterized model organisms for the study of C1 metabolism in methylotrophic bacteria, a diverse group of microbes that can use reduced one-carbon (C1) sources, such as methanol and methane as a sole source for both energy generation and carbon assimilation. Application of a match value (MV) based metric was used to rank the metabolite peaks in the data from those exhibiting the most mass spectral indications of labeling, to those not exhibiting any indications of labeling. The MV-based ranking corresponded well with analyst interpretation of the mass spectra. The MV-based method was initially demonstrated and validated using a mixture of 21 standards with data sets generated for mixtures at natural abundance, a mixture with 6 of the compounds labeled, and a 1:1 mixture of the natural abundance and labeled mixtures. Experimental data from TMS-derivatized extracts from the bacterium M. extorquens AM1 grown with natural abundance or (13)C-labeled methanol as the carbon source were analyzed. Of 131 peaks considered for the analysis of M. extorquens AM1, the 40 peaks ranked highest for indications of (13)C labeling were all found to be labeled, while those peaks ranked lower progressed from peaks for which labeling was uncertain, to a larger number of peaks that were clearly not labeled. The list of peaks determined to be labeled forms a library of compounds that are known to be labeled following the methanol metabolic pathway in M. extorquens AM1 that can be further investigated in future work, e.g. fluxomic studies. PMID:24007683

  3. Quantification of peptide m/z distributions from 13C-labeled cultures with high-resolution mass spectrometry.

    PubMed

    Allen, Doug K; Goldford, Joshua; Gierse, James K; Mandy, Dominic; Diepenbrock, Christine; Libourel, Igor G L

    2014-02-01

    Isotopic labeling studies of primary metabolism frequently utilize GC/MS to quantify (13)C in protein-hydrolyzed amino acids. During processing some amino acids are degraded, which reduces the size of the measurement set. The advent of high-resolution mass spectrometers provides a tool to assess molecular masses of peptides with great precision and accuracy and computationally infer information about labeling in amino acids. Amino acids that are isotopically labeled during metabolism result in labeled peptides that contain spatial and temporal information that is associated with the biosynthetic origin of the protein. The quantification of isotopic labeling in peptides can therefore provide an assessment of amino acid metabolism that is specific to subcellular, cellular, or temporal conditions. A high-resolution orbital trap was used to quantify isotope labeling in peptides that were obtained from unlabeled and isotopically labeled soybean embryos and Escherichia coli cultures. Standard deviations were determined by estimating the multinomial variance associated with each element of the m/z distribution. Using the estimated variance, quantification of the m/z distribution across multiple scans was achieved by a nonlinear fitting approach. Observed m/z distributions of uniformly labeled E. coli peptides indicated no significant differences between observed and simulated m/z distributions. Alternatively, amino acid m/z distributions obtained from GC/MS were convolved to simulate peptide m/z distributions but resulted in distinct profiles due to the production of protein prior to isotopic labeling. The results indicate that peptide mass isotopologue measurements faithfully represent mass distributions, are suitable for quantification of isotope-labeling-based studies, and provide additional information over existing methods. PMID:24387081

  4. Label free targeted detection and quantification of celiac disease immunogenic epitopes by mass spectrometry.

    PubMed

    van den Broeck, Hetty C; Cordewener, Jan H G; Nessen, Merel A; America, Antoine H P; van der Meer, Ingrid M

    2015-04-24

    Celiac disease (CD) is a food-related disease caused by certain gluten peptides containing T-cell stimulating epitopes from wheat, rye, and barley. CD-patients have to maintain a gluten-free diet and are therefore dependent on reliable testing and labeling of gluten-free products. So far, the R5-ELISA is the approved method to detect if food products can be labeled gluten-free. Because the R5-ELISA detects gluten in general, there is a demand for an improved detection method that quantifies specifically CD-epitopes. Therefore, we developed a new method for detection and quantification of CD-epitopes, based on liquid chromatography (LC) coupled to mass spectrometry (MS) in multiple reaction monitoring (MRM) mode. This method enables targeted label free comparative analysis of the gluten proteins present in different wheat varieties and species, and in wheat-based food products. We have tested our method by analyzing several wheat varieties that vary in CD-epitope content, as was shown before using immunoblotting and specific monoclonal antibodies. The results showed that a modern bread wheat variety Toronto contained the highest amounts of CD immunogenic peptides compared with the older bread wheat variety Minaret and the tetraploid wheat variety Dibillik Sinde. Our developed method can detect quantitatively and simultaneously multiple specific CD-epitopes in a high throughput manner. PMID:25795397

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  6. Kinetic folding mechanism of an integral membrane protein examined by pulsed oxidative labeling and mass spectrometry.

    PubMed

    Pan, Yan; Brown, Leonid; Konermann, Lars

    2011-07-01

    We report the application of pulsed oxidative labeling for deciphering the folding mechanism of a membrane protein. SDS-denatured bacteriorhodopsin (BR) was refolded by mixing with bicelles in the presence of free retinal. At various time points (20 ms to 1 day), the protein was exposed to a microsecond ·OH pulse that induces oxidative modifications at solvent-accessible methionine side chains. The extent of labeling was determined by mass spectrometry. These measurements were complemented by stopped-flow spectroscopy. Major time-dependent changes in solvent accessibility were detected for M20 (helix A) and M118 (helix D). Our kinetic data indicate a sequential folding mechanism, consistent with models previously suggested by others on the basis of optical data. Yet, ·OH labeling provides additional structural insights. An initial folding intermediate I(1) gets populated within 20 ms, concomitantly with formation of helix A. Subsequent structural consolidation leads to a transient species I(2). Noncovalent retinal binding to I(2) induces folding of helix D, thereby generating an intermediate I(R). In the absence of retinal, the latter transition does not take place. Hence, formation of helix D depends on retinal binding, whereas this is not the case for helix A. As the cofactor settles deeper into its binding pocket, a final transient species I(R) is generated. This intermediate converts into native BR within minutes by formation of the retinal-K216 Schiff base linkage. The combination of pulsed covalent labeling and optical spectroscopy employed here should also be suitable for exploring the folding mechanisms of other membrane proteins. PMID:21570983

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

    Jindal, S P; Lutz, T

    1989-12-01

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

  10. Direct high-spatial-resolution SIMS (secondary ion mass spectrometry) imaging of labeled nucleosides in human chromosomes

    NASA Astrophysics Data System (ADS)

    Hallegot, Philippe; Girod, C.; LeBeau, M. M.; Levi-Setti, Riccardo

    1991-03-01

    Using a scanning ion microprobe we analyzed the distribution of labelled thymidine along human chromosomes. Two labels have been used: bromodeoxyuridine (BrdU which contains one bromine atom per molecule) and 14C-thymidine (which contains either one or ten 14C atoms per molecule). Both types of labelled nucleosides can be detected with our insirument. Best results are obtained when using the uniformly labelled thymidine (U-14C-thymidine) and adding up in a KONTRON IBAS image processing system the sequential analytical maps acquired from the sample at mass 28 (14C14N ions). The distribution of thymidine is heterogeneous along the chromosomes and a banding pattern can be observed on the pictures (SIMS-bands). The spatial resolution obtained with our scanning ion microprobe (the University of Chicago Scanning Ion Microprobe: UC-SIM) surpasses the one of autoradiography which is the common direct method of localization of labelled nucleosides. 1.

  11. Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.

    PubMed

    Scheerlinck, E; Dhaenens, M; Van Soom, A; Peelman, L; De Sutter, P; Van Steendam, K; Deforce, D

    2015-12-01

    Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest. PMID:26302362

  12. Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method

    PubMed Central

    Maile, Tobias M.; Izrael-Tomasevic, Anita; Cheung, Tommy; Guler, Gulfem D.; Tindell, Charles; Masselot, Alexandre; Liang, Jun; Zhao, Feng; Trojer, Patrick; Classon, Marie; Arnott, David

    2015-01-01

    Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5. PMID:25680960

  13. Automated Label-free Quantification of Metabolites from Liquid Chromatography–Mass Spectrometry Data*

    PubMed Central

    Kenar, Erhan; Franken, Holger; Forcisi, Sara; Wörmann, Kilian; Häring, Hans-Ulrich; Lehmann, Rainer; Schmitt-Kopplin, Philippe; Zell, Andreas; Kohlbacher, Oliver

    2014-01-01

    Liquid chromatography coupled to mass spectrometry (LC-MS) has become a standard technology in metabolomics. In particular, label-free quantification based on LC-MS is easily amenable to large-scale studies and thus well suited to clinical metabolomics. Large-scale studies, however, require automated processing of the large and complex LC-MS datasets. We present a novel algorithm for the detection of mass traces and their aggregation into features (i.e. all signals caused by the same analyte species) that is computationally efficient and sensitive and that leads to reproducible quantification results. The algorithm is based on a sensitive detection of mass traces, which are then assembled into features based on mass-to-charge spacing, co-elution information, and a support vector machine–based classifier able to identify potential metabolite isotope patterns. The algorithm is not limited to metabolites but is applicable to a wide range of small molecules (e.g. lipidomics, peptidomics), as well as to other separation technologies. We assessed the algorithm's robustness with regard to varying noise levels on synthetic data and then validated the approach on experimental data investigating human plasma samples. We obtained excellent results in a fully automated data-processing pipeline with respect to both accuracy and reproducibility. Relative to state-of-the art algorithms, ours demonstrated increased precision and recall of the method. The algorithm is available as part of the open-source software package OpenMS and runs on all major operating systems. PMID:24176773

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

    PubMed

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

    1998-04-01

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

  15. A Bayesian Markov-Chain-Based Heteroscedastic Regression Model for the Analysis of 18O-Labeled Mass Spectra

    NASA Astrophysics Data System (ADS)

    Zhu, Qi; Burzykowski, Tomasz

    2011-03-01

    To reduce the influence of the between-spectra variability on the results of peptide quantification, one can consider the 18O-labeling approach. Ideally, with such labeling technique, a mass shift of 4 Da of the isotopic distributions of peptides from the labeled sample is induced, which allows one to distinguish the two samples and to quantify the relative abundance of the peptides. It is worth noting, however, that the presence of small quantities of 16O and 17O atoms during the labeling step can cause incomplete labeling. In practice, ignoring incomplete labeling may result in the biased estimation of the relative abundance of the peptide in the compared samples. A Markov model was developed to address this issue (Zhu, Valkenborg, Burzykowski. J. Proteome Res. 9, 2669-2677, 2010). The model assumed that the peak intensities were normally distributed with heteroscedasticity using a power-of-the-mean variance funtion. Such a dependence has been observed in practice. Alternatively, we formulate the model within the Bayesian framework. This opens the possibility to further extend the model by the inclusion of random effects that can be used to capture the biological/technical variability of the peptide abundance. The operational characteristics of the model were investigated by applications to real-life mass-spectrometry data sets and a simulation study.

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

  17. Preparation of fluorescent labeled gentamicin as biological tracer and its characterization by liquid chromatography and high resolution mass spectrometry.

    PubMed

    Woiwode, Ulrich; Sievers-Engler, Adrian; Lämmerhofer, Michael

    2016-03-20

    This work deals with the preparation of single-labeled bioconjugates of the antibiotic Gentamicin (GT) with the sulforhodamine-derived fluorescence dye Texas Red(®)-X (TR), its purification by high-performance liquid chromatography (HPLC) and its characterization by high-resolution mass spectrometry. Aminoglycosides such as GT are efficient antibiotics, but also problematic due to severe side effects such as nephro- and ototoxicity. Fluorescent labeled GT is used to visualize cellular uptake and distribution of the antibiotic to finally understand the mechanisms of serious adverse drug reactions. Pharmaceutically administered GT is a mixture of mainly four different components, which exhibit three (GT(C1)) or four (GT(C1a), GT(C2), GT(C2a)) primary amino functional groups which can be coupled with the labeling reagent TR. Thus, multiple labeling could be envisaged which was assumed to be detrimental for uptake studies by fluorescence imaging. The proposed synthesis aimed at preparation of single labeled product and together with the employed purification strategy indeed yielded single labeled GT as product. Analytical control of the reaction product was carried out by means of mass spectrometry (UHPLC-ESI-QTOF-MS/MS) to rule out over-labeling of GT, which would alter the physicochemical characteristics of GT and its cellular uptake significantly. Moreover, LC-MS/MS analysis gave valuable insights into structural diversity of single labeled products. Further, high-resolution MS and MS/MS spectra of underivatized GT are provided as well. The analytical information on preparation strategy and structure diversity is valuable for studies with a clinical focus on research of aminoglycoside toxicity. Furthermore, it is deemed to be useful for the development of LC-MS/MS assays for the determination of aminoglycosides or the fast screening of synthetic biology samples from biotechnological drug discovery. PMID:26775580

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

    PubMed Central

    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.

    2014-01-01

    We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (i) robust targeting of peptide N-termini and lysyl side chains, (ii) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (iii) 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 (iv) 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 extends the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers. PMID:24496597

  19. Protected amine labels: a versatile molecular scaffold for multiplexed nominal mass and sub-Da isotopologue quantitative proteomic reagents.

    PubMed

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

    2014-04-01

    We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers. PMID:24496597

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

    SciTech Connect

    Jewett, J.R., Fluor Daniel Hanford

    1997-02-24

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

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

    PubMed

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

    2015-08-01

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

  2. Rapid label-free determination of ketamine in whole blood using secondary ion mass spectrometry.

    PubMed

    Liao, Hua-Yang; Chen, Jung-Hsuan; Shyue, Jing-Jong; Shun, Chia-Tung; Chen, Huei-Wen; Liao, Su-Wei; Hong, Chih-Kang; Chen, Pai-Shan

    2015-10-01

    A fast and accurate drug screening to identify the possible presence of a wide variety of pharmaceutical and illicit drugs is increasingly requested in forensic and clinical toxicology. The current first-line screening relies on immunoassays. They determine only certain common drugs of which antibodies are commercially available. To address the issue, a rapid screening using secondary ion mass spectrometry (SIMS) has been developed. In the study, SIMS directly analyzed ketamine in whole blood without any pretreatment. While the untreated blood has a complicated composition, principal-components analysis (PCA) is used to detect unknown specimens by building up an analytical model from blank samples which were spiked with ketamine at 100 ng mL(-1), to simulate the presence of ketamine. Each characteristics m/z is normalized and scaled by multiplying the root square of intensity and square of corresponding m/z, developed by National Institute of Standards and Technology (NIST). Using linear regression and the result of PCA, this study enables to correctly distinguish ketamine positive and negative groups in an unknown set of specimens. The quantity of ketamine in an unknown set was determined using gas chromatography-mass spectrometry (GC-MS) as the reference methodology. Instead limited by commercially available antibodies, SIMS detects target molecules straight despite the label-free detection capabilities of SIMS, additional data processing (here, PCA) can be used to fully analyse the produced data, which extends the range of analytes of interest on drug screening. Furthermore, extremely low sample volume, 5 µL, is required owing to the high spatial resolution of SIMS. In addition, while the whole blood is analyzed within 3 min, the whole analysis has been shortened significantly and high throughput can be achieved. PMID:26078127

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

    PubMed

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

    2016-08-19

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

  4. Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

    PubMed Central

    Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18 kDa cationic antimicrobial protein (CAMP), and heat shock 70 kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC. PMID:25699276

  5. 18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

    SciTech Connect

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

    2011-10-11

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

  6. Combining combing and secondary ion mass spectrometry to study DNA on chips using (13)C and (15)N labeling.

    PubMed

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA - the combing, imaging by SIMS or CIS method - has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to (13)C-labeling via the detection and quantification of the (13)C (14)N (-) recombinant ion and the use of the (13)C: (12)C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  7. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling

    PubMed Central

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C 14N - recombinant ion and the use of the 13C: 12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  8. Modulating fluorescence anisotropy of dye-labeled DNA without involving mass amplification.

    PubMed

    Pei, Xiaojing; Huang, Hongduan; Chen, Yang; Li, Chenxi; Liu, Feng; Li, Na

    2016-07-01

    Fluorescence anisotropy, known as a simple, homogeneous and cost-effective analytical technology, is an invaluable technique for studying the micro-environmental changes of the dye associated with the molecular interactions. An in-depth understanding of the variables affecting the fluorescence anisotropy signal can facilitate better experimental designs to effectively improve the analytical performance. This work is a follow-up effort in evaluating the factors that can significantly influence fluorescence anisotropy. We systematically studied fluorescence anisotropy of dsDNA with the changing length based on dye-DNA interactions, with the fluorophores in the end-labeling, the middle-site-labeling, and multiple number of labeling manners. The fluorescence anisotropy value and the base-pair response dynamic range could be expanded by labeling the fluorophores in the middle of dsDNA and increasing the number of labels on dsDNA. The C overhang configuration in the end-labeling manner could enhance the fluorescence anisotropy signal but not expand the base-pair response range. Results from all the labeling fluorophores reinforced the leveling-off effect, i.e., the fluorescence anisotropy signal does not response to the increased length of the DNA duplex when the length is larger than a critical number of base pairs. These findings provide perspectives about choosing appropriate fluorescent dyes and labeling sites for simple and universal fluorescence anisotropy designs in various applications. PMID:27154716

  9. Measurement of deuterium-labeled phylloquinone in plasma by high-performance liquid chromatography/mass spectrometry.

    PubMed

    Fu, Xueyan; Peterson, James W; Hdeib, Mona; Booth, Sarah L; Grusak, Michael A; Lichtenstein, Alice H; Dolnikowski, Gregory G

    2009-07-01

    Phylloquinone (vitamin K(1)) is a lipophilic compound present in plasma at low concentrations, which presents technical challenges for determining its bioavailability or metabolic fate using stable isotopes. We developed a method to simultaneously measure unlabeled and deuterium-labeled phylloquinone concentrations in plasma specimens using high-performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC-APCI/MS). Phylloquinone was extracted from plasma using hexane, further purified by solid-phase extraction, and then quantified using high-performance liquid chromatography with an APCI/MS as a detector. Plotting the expected versus the measured amount of serial dilutions of either unlabeled or labeled phylloquinone gave correlation coefficients (R) of 0.999 for both compounds. The minimum detectable concentrations of unlabeled and labeled phylloquinone were 0.05 and 0.08 pmol/injection, respectively. Pooled plasma samples spiked with between 0.5 and 32 nmol phylloquinone/L gave average recoveries of 96.7% with 5.4% relative standard deviation (RSD) for unlabeled phylloquinone and 96.2% with 6.6% RSD for labeled phylloquinone. Plasma phylloquinone concentrations determined by LC-fluorescence and LC-APCI/MS methods from healthy subjects (n = 17) were not statistically different (P = 0.13). The LC-APCI/MS method is a sensitive technique for simultaneous determination of both unlabeled and labeled phylloquinone and can be applied to bioavailability studies. PMID:19563214

  10. Measurement of Deuterium-Labeled Phylloquinone in Plasma by High-Performance Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Fu, Xueyan; Peterson, James W.; Hdeib, Mona; Booth, Sarah L.; Grusak, Michael A.; Lichtenstein, Alice H.; Dolnikowski, Gregory G.

    2009-01-01

    Phylloquinone (vitamin K1) is a lipophilic compound present in plasma at low concentrations, which presents technical challenges for determining its bioavailability or metabolic fate using stable isotopes. We developed a method to simultaneously measure unlabeled and deuterium-labeled phylloquinone concentrations in plasma specimens using high-performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC–APCI/MS). Phylloquinone was extracted from plasma using hexane, further purified by solid-phase extraction, and then quantified using high-performance liquid chromatography with an APCI/MS as a detector. Plotting the expected versus the measured amount of serial dilutions of either unlabeled or labeled phylloquinone gave correlation coefficients (R) of 0.999 for both compounds. The minimum detectable concentrations of unlabeled and labeled phylloquinone were 0.05 and 0.08 pmol/injection, respectively. Pooled plasma samples spiked with between 0.5 and 32 nmol phylloquinone/L gave average recoveries of 96.7% with 5.4% relative standard deviation (RSD) for unlabeled phylloquinone and 96.2% with 6.6% RSD for labeled phylloquinone. Plasma phylloquinone concentrations determined by LC-fluorescence and LC–APCI/MS methods from healthy subjects (n = 17) were not statistically different (P = 0.13). The LC–PCI/MS method is a sensitive technique for simultaneous determination of both unlabeled and labeled phylloquinone and can be applied to bioavailability studies. PMID:19563214

  11. Bayesian identification of protein differential expression in multi-group isobaric labelled mass spectrometry data.

    PubMed

    Jow, Howsun; Boys, Richard J; Wilkinson, Darren J

    2014-10-01

    In this paper we develop a Bayesian statistical inference approach to the unified analysis of isobaric labelled MS/MS proteomic data across multiple experiments. An explicit probabilistic model of the log-intensity of the isobaric labels' reporter ions across multiple pre-defined groups and experiments is developed. This is then used to develop a full Bayesian statistical methodology for the identification of differentially expressed proteins, with respect to a control group, across multiple groups and experiments. This methodology is implemented and then evaluated on simulated data and on two model experimental datasets (for which the differentially expressed proteins are known) that use a TMT labelling protocol. PMID:25153608

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2008-10-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

    Cox, Jürgen

    2016-01-01

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

  16. Mass Spectral Characterization of Organophosphate-Labeled, Tyrosine-Containing Peptides: Characteristic Mass Fragments and A New Binding Motif for Organophosphates

    PubMed Central

    Schopfer, Lawrence M.; Grigoryan, Hasmik; Li, Bin; Nachon, Florian; Masson, Patrick; Lockridge, Oksana

    2009-01-01

    We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with 6 different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8–8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the present work were 1) to determine the common features of the OP-reactive tyrosines, and 2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 amu and 244 amu for tyrosine-OP immonium ions were nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos oxon. Characteristic fragments also appeared from the parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to find when the OP bears no radiolabel and no tag. The characteristic MSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide. PMID:19762289

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

    SciTech Connect

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

    1990-10-01

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

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

    PubMed

    McConnell, Brian O; Antoniewicz, Maciek R

    2016-05-01

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

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

    PubMed

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

    2005-01-01

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

  20. 76 FR 82129 - Medical Devices; Ovarian Adnexal Mass Assessment Score Test System; Labeling; Black Box Restrictions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-30

    ... surgery. In the Federal Register of March 23, 2011 (76 FR 16292 at 12694), FDA published a final rule that... black box warning to address the risk of off-label use. In the Federal Register of March 23, 2011 (76 FR... Federal Register of March 23, 2011 (76 FR 16350 at 16352), FDA published a proposed rule to require...

  1. 76 FR 16350 - Medical Devices; Ovarian Adnexal Mass Assessment Score Test System; Labeling; Black Box Restrictions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-23

    ... HUMAN SERVICES Food and Drug Administration 21 CFR Part 866 Medical Devices; Ovarian Adnexal Mass... regulation classifying ovarian adnexal mass assessment score test systems to restrict these devices so that a... mass assessment score test system into class II (special controls). DATES: Submit either electronic...

  2. Segmentation of precursor mass range using "tiling" approach increases peptide identifications for MS1-based label-free quantification.

    PubMed

    Vincent, Catherine E; Potts, Gregory K; Ulbrich, Arne; Westphall, Michael S; Atwood, James A; Coon, Joshua J; Weatherly, D Brent

    2013-03-01

    Label-free quantification is a powerful tool for the measurement of protein abundances by mass spectrometric methods. To maximize quantifiable identifications, MS(1)-based methods must balance the collection of survey scans and fragmentation spectra while maintaining reproducible extracted ion chromatograms (XIC). Here we present a method which increases the depth of proteome coverage over replicate data-dependent experiments without the requirement of additional instrument time or sample prefractionation. Sampling depth is increased by restricting precursor selection to a fraction of the full MS(1) mass range for each replicate; collectively, the m/z segments of all replicates encompass the full MS(1) range. Although selection windows are narrowed, full MS(1) spectra are obtained throughout the method, enabling the collection of full mass range MS(1) chromatograms such that label-free quantitation can be performed for any peptide in any experiment. We term this approach "binning" or "tiling" depending on the type of m/z window utilized. By combining the data obtained from each segment, we find that this approach increases the number of quantifiable yeast peptides and proteins by 31% and 52%, respectively, when compared to normal data-dependent experiments performed in replicate. PMID:23350991

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

    PubMed

    Zhou, Ruokun; Li, Liang

    2014-01-01

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

  4. Structural Insights into the Pre-amyloid Tetramer of β-2-microglobulin from Covalent Labeling and Mass Spectrometry‡

    PubMed Central

    Mendoza, Vanessa Leah; Barón-Rodríguez, Mario A.; Blanco, Cristian; Vachet, Richard W.

    2011-01-01

    The main pathogenic process underlying dialysis-related amyloidosis (DRA) is the accumulation of β-2-microglobulin (β2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β2m amyloid formation. Cu(II)-induced β2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type β2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. This covalent labeling data along with molecular dynamics calculations enable the construction of a tetramer model that indicates how the protein might proceed to form even higher order oligomers. PMID:21718071

  5. Structural characterization of an integral membrane protein in its natural lipid environment by oxidative methionine labeling and mass spectrometry.

    PubMed

    Pan, Yan; Stocks, Bradley B; Brown, Leonid; Konermann, Lars

    2009-01-01

    Membrane proteins represent formidable challenges for many analytical techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a non-natural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-containing purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dilute aqueous H(2)O(2). The experiments rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following .OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-soluble proteins, which generally undergo modifications at many different types of residues. In those earlier experiments the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidation pattern observed here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidation. Our results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, especially when considering that many of these species are methionine-rich. The introduction of additional Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodology described here. PMID:19055344

  6. Human Vitamin B12 Absorption and Metabolism are Measured by Accelerator Mass Spectrometry Using Specifically Labeled 14C-Cobalamin

    SciTech Connect

    Carkeet, C; Dueker, S R; Lango, J; Buchholz, B A; Miller, J W; Green, R; Hammock, B D; Roth, J R; Anderson, P J

    2006-01-26

    There is need for an improved test of human ability to assimilate dietary vitamin B{sub 12}. Assaying and understanding absorption and uptake of B{sub 12} is important because defects can lead to hematological and neurological complications. Accelerator mass spectrometry (AMS) is uniquely suited for assessing absorption and kinetics of {sup 14}C-labeled substances after oral ingestion because it is more sensitive than decay counting and can measure levels of carbon-14 ({sup 14}C) in microliter volumes of biological samples, with negligible exposure of subjects to radioactivity. The test we describe employs amounts of B{sub 12} in the range of normal dietary intake. The B{sub 12} used was quantitatively labeled with {sup 14}C at one particular atom of the DMB moiety by exploiting idiosyncrasies of Salmonellametabolism. In order to grow aerobically on ethanolamine, S. entericamust be provided with either pre-formed B{sub 12} or two of its precursors: cobinamide and dimethylbenzimidazole (DMB). When provided with {sup 14}C-DMB specifically labeled in the C2 position, cells produced {sup 14}C-B{sub 12} of high specific activity (2.1 GBq/mmol, 58 mCi/mmol) and no detectable dilution of label from endogenous DMB synthesis. In a human kinetic study, a physiological dose (1.5 mg, 2.2 KBq/59 nCi) of purified {sup 14}C-B{sub 12} was administered and showed plasma appearance and clearance curves consistent with the predicted behavior of the pure vitamin. This method opens new avenues for study of B{sub 12} assimilation.

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

    PubMed

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

    2016-05-01

    This study presents a new chemical cross-linking mass spectrometry (MS) method in combination with electrochemistry and isotope labeling strategy for probing both protein three-dimensional (3D) structures and conformational changes. For the former purpose, the target protein/protein complex is cross-linked with equal mole of premixed light and heavy isotope labeled cross-linkers carrying electrochemically reducible disulfide bonds (i.e., DSP-d0 and DSP-d8 in this study, DSP = dithiobis[succinimidyl propionate]), digested and then electrochemically reduced followed with online MS analysis. Cross-links can be quickly identified because of their reduced intensities upon electrolysis and the presence of doublet isotopic peak characteristics. In addition, electroreduction converts cross-links into linear peptides, facilitating MS/MS analysis to gain increased information about their sequences and modification sites. For the latter purpose of probing protein conformational changes, an altered procedure is adopted, in which the protein in two different conformations is cross-linked using DSP-d0 and DSP-d8 separately, and then the two protein samples are mixed in 1:1 molar ratio. The merged sample is subjected to digestion and electrochemical mass spectrometric analysis. In such a comparative cross-linking experiment, cross-links could still be rapidly recognized based on their responses to electrolysis. More importantly, the ion intensity ratios of light and heavy isotope labeled cross-links reveal the conformational changes of the protein, as exemplified by examining the effect of Ca(2+) on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology. Graphical Abstract ᅟ. PMID:26902947

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-15

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

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

    DOE PAGESBeta

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

    2008-01-01

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

  14. Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously.

    PubMed

    Blank, Lars M; Desphande, Rahul R; Schmid, Andreas; Hayen, Heiko

    2012-06-01

    Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously--i.e., (13)C and (15)N--in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with (13)C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon-nitrogen co-metabolism by administering both (13)C-labeled glucose and (15)N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes. PMID:22543713

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

    PubMed

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

    2016-03-01

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

  16. Production, Purification, and Characterization of 15N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements

    PubMed Central

    Jaruga, Pawel; Nelson, Bryant C.; Lowenthal, Mark S.; Jemth, Ann-Sofie; Loseva, Olga; Coskun, Erdem; Helleday, Thomas

    2016-01-01

    Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA–protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length 15N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of 15N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented. PMID:26791985

  17. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

    PubMed

    Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

  18. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine

    PubMed Central

    Dick Jr, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    ABSTRACT The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90–110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

  19. Dynamic Changes in Rat Mesenteric Lymph Proteins Following Trauma Using Label-Free Mass Spectrometry

    PubMed Central

    D’Alessandro, Angelo; Dzieciatkowska, Monika; Peltz, Erik D.; Moore, Ernest E.; Jordan, Janeen R.; Silliman, Christopher C.; Banerjee, Anirban; Hansen, Kirk C.

    2014-01-01

    Early events triggered by post-trauma/hemorrhagic shock currently represent a leading cause of morbidity and mortality in these patients. The causative agents of these events have been associated with increased neutrophil priming secondary to shock-dependent alterations of mesenteric lymph. Previous studies have suggested that unknown soluble components of the post-shock mesenteric lymph are main drivers of these events. In the present study, we applied a label free proteomics approach to further delve into the early proteome changes of the mesenteric lymph in response to hemorrhagic shock. Time-course analyses were performed by sampling the lymph every thirty minutes post-shock up until 3h (the time window within which a climax in neutrophil priming was observed). There are novel, transient early post-hemorrhagic shock alterations to the proteome and previously undocumented post-shock protein alterations. These results underlie the triggering of coagulation and pro-inflammatory responses secondary to trauma/hemorrhagic shock, metabolic deregulation and apoptosis, and alterations to proteases/anti-proteases homeostasis, which are suggestive of the potential implication of extracellular matrix proteases in priming neutrophil activation. Finally, there is a likely correlation between early PSML post-shock neutrophil priming and proteomics changes, above all protease/anti-proteases impaired homeostasis (especially of serine proteases and metalloproteases). PMID:25243424

  20. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    PubMed

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  1. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    PubMed Central

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  2. Residual CTAB Ligands as Mass Spectrometry Labels to Monitor Cellular Uptake of Au Nanorods.

    PubMed

    García, Isabel; Henriksen-Lacey, Malou; Sánchez-Iglesias, Ana; Grzelczak, Marek; Penadés, Soledad; Liz-Marzán, Luis M

    2015-06-01

    Gold nanorods have numerous applications in biomedical research, including diagnostics, bioimaging, and photothermal therapy. Even though surfactant removal and surface conjugation with antifouling molecules such as polyethylene glycol (PEG) are required to minimize nonspecific protein binding and cell uptake, the reliable characterization of these processes remains challenging. We propose here the use of laser desorption/ionization mass spectrometry (LDI-MS) to study the ligand exchange efficiency of cetyltrimethylammonium bromide (CTAB)-coated nanorods with different PEG grafting densities and to characterize nanorod internalization in cells. Application of LDI-MS analysis shows that residual CTAB consistently remains adsorbed on PEG-capped Au nanorods. Interestingly, such residual CTAB can be exploited as a mass barcode to discern the presence of nanorods in complex fluids and in vitro cellular systems, even at very low concentrations. PMID:26266492

  3. ¹³C labelled internal standards--a solution to minimize ion suppression effects in liquid chromatography-tandem mass spectrometry analyses of drugs in biological samples?

    PubMed

    Berg, Thomas; Strand, Dag Helge

    2011-12-30

    Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is frequently used to identify and quantify drugs in human biological samples due to the high selectivity and sensitivity of this technique. However, ion suppression effects caused by co-eluting compounds: drugs, metabolites, matrix components, impurities and degradation products, are a major concern. Stable isotope labelled internal standards (SIL ISs), usually deuterium ((2)H) labelled, are often used to compensate for these effects. In many LC separations the retention times of (2)H labelled ISs and their analogues will differ. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is increasingly being used for bio-analysis. With the better chromatographic resolution provided with sub 2 μm particles, larger separation between analytes and their (2)H labelled analogues can be expected, which might reduce the benefits of the SIL IS. There is a greater difference in physico-chemical properties between hydrogen isotopes than between isotopes of other elements. (13)C, (15)N and (18)O labelled ISs are more similar to their analytes than (2)H labelled ISs and thereby expected to behave more similarly in chromatographic separations. In this study we have investigated the use of (13)C and (2)H labelled ISs for the determination of amphetamine and methamphetamine by UPLC-MS/MS. The (13)C labelled ISs were co eluting with their analytes under different chromatographic conditions while the (2)H labelled ISs and their analytes were slightly separated. An improved ability to compensate for ion suppression effects were observed when the (13)C labelled ISs were used. Furthermore, an UPLC-MS/MS method for determination of amphetamine and methamphetamine in urine using (13)C labelled ISs has been developed and validated. Unfortunately, there are few (13)C labelled ISs commercial available today. If more (13)C labelled ISs become commercial available they may well be the coming solution to minimize

  4. Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry.

    PubMed

    Yuan, Jiangbei; Wang, Chengjian; Sun, Yujiao; Huang, Linjuan; Wang, Zhongfu

    2014-10-01

    A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate-peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies. PMID:24912132

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    2011-01-01

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

  7. Constructing Proteome Reference Map of the Porcine Jejunal Cell Line (IPEC-J2) by Label-Free Mass Spectrometry.

    PubMed

    Kim, Sang Hoon; Pajarillo, Edward Alain B; Balolong, Marilen P; Lee, Ji Yoon; Kang, Dae-Kyung

    2016-06-28

    In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications. PMID:26975772

  8. A new approach for the comparative analysis of multiprotein complexes based on 15N metabolic labeling and quantitative mass spectrometry.

    PubMed

    Trompelt, Kerstin; Steinbeck, Janina; Terashima, Mia; Hippler, Michael

    2014-01-01

    The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling ((14)N/(15)N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions. PMID:24686495

  9. Label free biochemical 2D and 3D imaging using secondary ion mass spectrometry

    PubMed Central

    Fletcher, John S.; Vickerman, John C.; Winograd, Nicholas

    2011-01-01

    Time-of-flight Secondary ion mass spectrometry (ToF-SIMS) provides a method for the detection of native and exogenous compounds in biological samples on a cellular scale. Through the development of novel ion beams the amount of molecular signal available from the sample surface has been increased. Through the introduction of polyatomic ion beams, particularly C60, ToF-SIMS can now be used to monitor molecular signals as a function of depth as the sample is eroded thus proving the ability to generate 3D molecular images. Here we describe how this new capability has led to the development of novel instrumentation for 3D molecular imaging while also highlighting the importance of sample preparation and discuss the challenges that still need to be overcome to maximise the impact of the technique. PMID:21664172

  10. Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

    NASA Astrophysics Data System (ADS)

    Smeekens, Johanna M.; Chen, Weixuan; Wu, Ronghu

    2015-04-01

    Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide- N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

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

    PubMed

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

    2013-09-01

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

  12. Normalization Approaches for Removing Systematic Biases Associated with Mass Spectrometry and Label-Free Proteomics

    SciTech Connect

    Callister, Stephen J.; Barry, Richard C.; Adkins, Joshua N.; Johnson, Ethan T.; Qian, Weijun; Webb-Robertson, Bobbie-Jo M.; Smith, Richard D.; Lipton, Mary S.

    2006-02-01

    Central tendency, linear regression, locally weighted regression, and quantile techniques were investigated for normalization of peptide abundance measurements obtained from high-throughput liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS). Arbitrary abundances of peptides were obtained from three sample sets, including a standard protein sample, two Deinococcus radiodurans samples taken from different growth phases, and two mouse striatum samples from control and methamphetamine-stressed mice (strain C57BL/6). The selected normalization techniques were evaluated in both the absence and presence of biological variability by estimating extraneous variability prior to and following normalization. Prior to normalization, replicate runs from each sample set were observed to be statistically different, while following normalization replicate runs were no longer statistically different. Although all techniques reduced systematic bias, assigned ranks among the techniques revealed significant trends. For most LC-FTICR MS analyses, linear regression normalization ranked either first or second among the four techniques, suggesting that this technique was more generally suitable for reducing systematic biases.

  13. MASS LOSS AND NITROGEN DYNAMICS DURING THE DECOMPOSITION OF A N-LABELED N2-FIXING EPOPHYTIC LICHEN, LOBARIA OREGANA (TUCK.) MULL. ARG.

    EPA Science Inventory

    We studied mass loss and nitrogen dynamics during fall and spring initiated decomposition of an N2-fixing epiphytic lichen, Lobaria oregana (Tuck.) Mull. Arg. using 15N. We developed a method of labeling lichens with 15N that involved spraying lichen material with a nutrient sol...

  14. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    PubMed Central

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

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

    PubMed

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

    2015-04-01

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

  16. Label-Free, In-Solution Screening of Peptide Libraries for Binding to Protein Targets Using Hydrogen Exchange Mass Spectrometry.

    PubMed

    Maaty, Walid S; Weis, David D

    2016-02-01

    There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g., phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange is measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the Escherichia coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494-513) and a peptide spanning the N-terminal 20 residues of bovine ribonuclease A (S peptide). Human calmodulin and bovine ribonuclease S (RNase S) were screened against the library. Using a novel data analysis workflow, we identified the eNOS peptide as the only calmodulin binding peptide and S peptide as the only ribonuclease S binding peptide in the library. PMID:26741284

  17. Characterization of the MDSC proteome associated with metastatic murine mammary tumors using label-free mass spectrometry and shotgun proteomics.

    PubMed

    Boutté, Angela M; McDonald, W Hayes; Shyr, Yu; Yang, Li; Lin, P Charles

    2011-01-01

    Expansion of Gr-1+/CD11b+ myeloid derived suppressor cells (MDSCs) is governed by the presence of increasingly metastatic, malignant primary tumors. Metastasis, not the primary tumor, is often the cause of mortality. This study sought to fully characterize the MDSC proteome in response to metastatic and non-metastatic mammary tumors using label-free mass spectrometry shotgun proteomics in a mouse model with tumor cell lines, 67NR and 4T1, derived from the same tumor. 67NR cells form only primary mammary tumors, whereas 4T1 cells readily metastasize to the lungs, lymph nodes, and blood. Overall analysis identified a total of 2825 protein groups with a 0.78% false discovery rate. Of the 2814 true identifications, 43 proteins were exclusive to the 67NR group, 153 were exclusive to the 4T1 group, and 2618 were shared. Among the shared cohort, 26 proteins were increased and 31 were decreased in the metastatic 4T1 cohort compared to non-metastatic 67NR controls after filtering. MDSCs selectively express proteins involved in the γ-glutamyl transferase, glutathione synthase pathways, CREB transcription factor signaling, and other pathways involved in platelet aggregation, as well as lipid and amino acid metabolism, in response to highly metastatic 4T1 tumors. Cell cycle regulation dominated protein pathways and ontological groups of the 67NR non-metastatic group. Not only does this study provide a starting point to identify potential biomarkers of metastasis expressed by MDSCs; it identifies critical pathways that are unique to non-metastatic and metastatic conditions. Therapeutic interventions aimed at these pathways in MDSC may offer a new route to control malignancy and metastasis. PMID:21853032

  18. Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass Spectrometry*

    PubMed Central

    Manes, Nathan P.; Dong, Li; Zhou, Weidong; Du, Xiuxia; Reghu, Nikitha; Kool, Arjan C.; Choi, Dahan; Bailey, Charles L.; Petricoin, Emanuel F.; Liotta, Lance A.; Popov, Serguei G.

    2011-01-01

    Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24–48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48–72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic

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

    SciTech Connect

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

    1988-03-01

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

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

    PubMed Central

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

    2010-01-01

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

  1. Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using O-16/O-18 Labeling and the Accurate Mass and Time Tag Approach

    SciTech Connect

    Qian, Weijun; Monroe, Matthew E.; Liu, Tao; Jacobs, Jon M.; Anderson, Gordon A.; Shen, Yufeng; Moore, Ronald J.; Anderson, David J.; Zhang, Rui; Calvano, Steven E.; Lowry, Stephen F.; Xiao, Wenzhong; Moldawer, Lyle L.; Davis, Ronald W.; Tompkins, Ronald G.; Camp, David G.; Smith, Richard D.

    2005-05-01

    Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. We describe here an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes post-digestion trypsin-catalyzed 16O/18O labeling, two-dimensional liquid chromatography (LC)-Fourier transform ion cyclotron resonance ((FTICR) mass spectrometry, and the accurate mass and time (AMT) tag strategy for identification and quantification of peptides/proteins from complex samples. A peptide mass and time tag database was initially generated using tandem mass spectrometry (MS/MS) following extensive multidimensional LC separations and the database serves as a ‘look-up’ table for peptide identification. The mass and time tag database contains >8,000 putative identified peptides, which yielded 938 confident plasma protein identifications. The quantitative approach was applied to the comparative analyses of plasma samples from an individual prior to and 9 hours after lipopolysaccharide (LPS) administration without depletion of high abundant proteins. Accurate quantification of changes in protein abundance was demonstrated with both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses and the protein abundances for 28 proteins were observed to be significantly changed following LPS administration, including several known inflammatory response mediators.

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2016-01-01

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

  4. Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by 13C-/12C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

    Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by 13C-dansyl and 12C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

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

    PubMed

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

    2016-02-18

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2013-01-01

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

  10. The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham.

    PubMed

    Gallego, Marta; Mora, Leticia; Concepción Aristoy, M; Toldrá, Fidel

    2016-04-01

    The aim of this work was to quantify changes in the abundance of the major sarcoplasmic proteins throughout the ham dry-curing process by using a label-free mass spectrometry methodology based on the measurement of mass spectral peak intensities obtained from the extracted ion chromatogram. For this purpose, extraction of sarcoplasmic proteins was followed by trypsin digestion and analysis by nanoliquid chromatography coupled to tandem mass spectrometry (Q/TOF) for the identification and relative quantification of sarcoplasmic proteins through individual quantification of trypsinised peptides. In total, 20 proteins, including 12 glycolytic enzymes, were identified and quantified. The accuracy of the protocol was based on MS/MS replicates, and beta-lactoglobulin protein was used to normalise data and correct possible variations during sample preparation or LC-MS/MS analysis. Mass spectrometry-based proteomics provides precise identification and quantification of proteins in comparison with traditional methodologies based on gel electrophoresis, especially in the case of overlapping proteins. Moreover, the label-free approach used in this study proved to be a simple, fast, reliable method for evaluating proteolytic degradation of sarcoplasmic proteins during the processing of dry-cured ham. PMID:26593512

  11. Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using 16O/18O Labeling and the Accurate Mass and Time Tag Approach

    PubMed Central

    Qian, Wei-Jun; Monroe, Matthew E.; Liu, Tao; Jacobs, Jon M.; Anderson, Gordon A.; Shen, Yufeng; Moore, Ronald J.; Anderson, David J.; Zhang, Rui; Calvano, Steve E.; Lowry, Stephen F.; Xiao, Wenzhong; Moldawer, Lyle L.; Davis, Ronald W.; Tompkins, Ronald G.; Camp, David G.; Smith, Richard D.

    2007-01-01

    SUMMARY Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes post-digestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional liquid chromatography (LC)-Fourier transform ion cyclotron resonance ((FTICR) mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC-elution time AMT tag database was initially generated using tandem mass spectrometry (MS/MS) following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag database contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion for high abundant proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration. PMID:15753121

  12. Exemplifying the Screening Power of Mass Spectrometry Imaging over Label-Based Technologies for Simultaneous Monitoring of Drug and Metabolite Distributions in Tissue Sections.

    PubMed

    Goodwin, Richard J A; Nilsson, Anna; Mackay, C Logan; Swales, John G; Johansson, Maria K; Billger, Martin; Andrén, Per E; Iverson, Suzanne L

    2016-02-01

    Mass spectrometry imaging (MSI) provides pharmaceutical researchers with a suite of technologies to screen and assess compound distributions and relative abundances directly from tissue sections and offer insight into drug discovery-applicable queries such as blood-brain barrier access, tumor penetration/retention, and compound toxicity related to drug retention in specific organs/cell types. Label-free MSI offers advantages over label-based assays, such as quantitative whole-body autoradiography (QWBA), in the ability to simultaneously differentiate and monitor both drug and drug metabolites. Such discrimination is not possible by label-based assays if a drug metabolite still contains the radiolabel. Here, we present data exemplifying the advantages of MSI analysis. Data of the distribution of AZD2820, a therapeutic cyclic peptide, are related to corresponding QWBA data. Distribution of AZD2820 and two metabolites is achieved by MSI, which [(14)C]AZD2820 QWBA fails to differentiate. Furthermore, the high mass-resolving power of Fourier transform ion cyclotron resonance MS is used to separate closely associated ions. PMID:26701101

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

    PubMed

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

    2016-05-01

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

  14. Use of 4-sulfophenyl isothiocyanate labeling and mass spectrometry to determine the site of action of the streptococcolytic peptidoglycan hydrolase zoocin A.

    PubMed

    Gargis, Shaw R; Heath, Harry E; Heath, Lucie S; Leblanc, Paul A; Simmonds, Robin S; Abbott, Brian D; Timkovich, Russell; Sloan, Gary L

    2009-01-01

    Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a d-alanyl-l-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases. PMID:18978086

  15. Differential Label-free Quantitative Proteomic Analysis of Shewanella oneidensis Cultured under Aerobic and Suboxic Conditions by Accurate Mass and Time Tag Approach

    SciTech Connect

    Fang, Ruihua; Elias, Dwayne A.; Monroe, Matthew E.; Shen, Yufeng; McIntosh, Martin; Wang, Pei; Goddard, Carrie D.; Callister, Stephen J.; Moore, Ronald J.; Gorby, Yuri A.; Adkins, Joshua N.; Fredrickson, Jim K.; Lipton, Mary S.; Smith, Richard D.

    2006-04-01

    We describe the application of liquid chromatography coupled to mass spectrometry (LC/MS) without the use of stable isotope labeling for differential quantitative proteomics analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and sub-oxic conditions. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to initially identify peptide sequences, and LC coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) was used to confirm these identifications, as well as measure relative peptide abundances. 2343 peptides, covering 668 proteins were identified with high confidence and quantified. Among these proteins, a subset of 56 changed significantly using statistical approaches such as SAM, while another subset of 56 that were annotated as performing housekeeping functions remained essentially unchanged in relative abundance. Numerous proteins involved in anaerobic energy metabolism exhibited up to a 10-fold increase in relative abundance when S. oneidensis is transitioned from aerobic to sub-oxic conditions.

  16. Measurement of deuterium-labeled phylloquinone in plasma by high-performance liquid chromatography/mass spectrometry (LC/MS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phylloquinone (vitamin K1) is a lipophilic compound present in plasma at low concentrations, which presents technical challenges for determining its bioavailability or metabolic fate using stable isotopes. We developed a method to simultaneously measure unlabeled and deuterium-labeled phylloquinone ...

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

    PubMed

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

    2016-03-01

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

  18. Segmentation of precursor mass range using ‘tiling’ approach increases peptide identifications for MS1-based label-free quantification

    PubMed Central

    Vincent, Catherine E.; Potts, Gregory K.; Ulbrich, Arne; Westphall, Michael S.; Atwood, James A.; Coon, Joshua J.; Weatherly, D. Brent

    2013-01-01

    Label-free quantification is a powerful tool for the measurement of protein abundances by mass spectrometric methods. To maximize quantifiable identifications, MS1-based methods must balance the collection of survey scans and fragmentation spectra while maintaining reproducible extracted ion chromatograms (XIC). Here we present a method which increases the depth of proteome coverage over replicate data-dependent experiments without the requirement of additional instrument time or sample pre-fractionation. Sampling depth is increased by restricting precursor selection to a fraction of the full MS1 mass range for each replicate; collectively, the m/z segments of all replicates encompass the full MS1 range. Although selection windows are narrowed, full MS1 spectra are obtained throughout the method, enabling the collection of full mass range MS1 chromatograms such that label-free quantitation can be performed for any peptide in any experiment. We term this approach “binning” or “tiling” depending on the type of m/z window utilized. By combining the data obtained from each segment, we find that this approach increases the number of quantifiable yeast peptides and proteins by 31% and 52%, respectively, when compared to normal data-dependent experiments performed in replicate. PMID:23350991

  19. Tc-99m-labeled red blood cells for the measurement of red cell mass in newborn infants: concise communication

    SciTech Connect

    Linderkamp, O.; Betke, K.; Fendel, H.; Klemm, J.; Lorenzen, K.; Riegel, K.P.

    1980-07-01

    In vitro and in vivo investigations were performed to examine the binding of Tc-99m to neonatal red blood cells (RBC). Labeling efficiency was about 90%, and unbound Tc-99m less than 3% after one washing, in premature and full-term newborns and in children. Thus presence of high percentages of fetal hemoglobin (Hb F) did not influence the labeling of RBCs with Tc-99m. RBCs of 11 newborns were hemolysed and the distribution of Tc-99m on RBC components was analyzed. Although Hb F percentage averaged (60.0 +- 8.10)% (s.d.), only (11.9 +- 3.7)% of Tc-99m was bound by Hb F, whereas (45.0 +- 6.1)% was associated with Hb A. RBC membranes bound (13.7 +- 4.3)% and (29.3 +- 4.0)% were found unbound in hemolysates. These results indicate that Tc-99m preferentially binds to beta chains. In vivo equilibration of Tc-99m RBCs and of albumin labeled with Evans blue was investigated in five newborn infants. Tc-99m RBCs were stable in each case during the first hour after injection. Elution of Tc-99m from RBCs was (3.4 +- 1.5)% per h. Body-to-venous hematocrit ratio averaged 0.86 +- 0.03.

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

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

    PubMed

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

    2014-10-01

    Because of a limited volume of urine that can be collected from a mouse, it is very difficult to apply the common strategy of using multiple analytical techniques to analyze the metabolites to increase the metabolome coverage for mouse urine metabolomics. We report an enabling method based on differential isotope labeling liquid chromatography mass spectrometry (LC-MS) for relative quantification of over 950 putative metabolites using 20 μL of urine as the starting material. The workflow involves aliquoting 10 μL of an individual urine sample for ¹²C-dansylation labeling that target amines and phenols. Another 10 μL of aliquot was taken from each sample to generate a pooled sample that was subjected to ¹³C-dansylation labeling. The ¹²C-labeled individual sample was mixed with an equal volume of the ¹³C-labeled pooled sample. The mixture was then analyzed by LC-MS to generate information on metabolite concentration differences among different individual samples. The interday repeatability for the LC-MS runs was assessed, and the median relative standard deviation over 4 days was 5.0%. This workflow was then applied to a metabolomic biomarker discovery study using urine samples obtained from the TgCRND8 mouse model of early onset familial Alzheimer's disease (FAD) throughout the course of their pathological deposition of beta amyloid (Aβ). It was showed that there was a distinct metabolomic separation between the AD prone mice and the wild type (control) group. As early as 15-17 weeks of age (presymptomatic), metabolomic differences were observed between the two groups, and after the age of 25 weeks the metabolomic alterations became more pronounced. The metabolomic changes at different ages corroborated well with the phenotype changes in this transgenic mice model. Several useful candidate biomarkers including methionine, desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic acid were identified. Some of them were found in previous

  2. Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global 13C-labeled internal standards improve performance for quantitative metabolomics in bacteria

    PubMed Central

    Yang, Song; Sadilek, Martin; Lidstrom, Mary E.

    2010-01-01

    Streamlined quantitative metabolomics in central metabolism of bacteria would be greatly facilitated by a high-efficiency liquid chromatography (LC) method in conjunction with accurate quantitation. To achieve this goal, a methodology for LC-tandem quadrupole mass spectrometry (LC-MS/MS) involving a pentafluorophenylpropyl (PFPP) column and culture-derived global 13C-labeled internal standards (I.Ss.) has been developed and compared to hydrophilic interaction liquid chromatography (HILIC)-MS/MS and published combined two-dimensional gas chromatography and LC methods. All 50 tested metabolite standards from 5 classes (amino acids, carboxylic acids, nucleotides, acyl-CoAs and sugar phosphates) displayed good chromatographic separation and sensitivity on the PFPP column. In addition, many important critical pairs such as isomers / isobars (e.g. isoleucine / leucine, methylsuccinic acid / ethylmalonic acid and malonyl-CoA / 3-hydroxybutyryl-CoA) and metabolites of similar structure (e.g. malate / fumarate) were resolved better on the PFPP than on the HILIC column. Compared to only one 13C-labeled I.S., the addition of global 13C-labeled I.Ss. improved quantitative linearity and accuracy. PFPP-MS/MS with global 13C-labeled I.Ss. allowed the absolute quantitation of 42 metabolite pool sizes in M. extorquens AM1. A comparison of metabolite level changes published previously for ethylamine (C2) versus succinate (C4) cultures of Methylobacterium extorquens AM1 indicated a good consistency with the data obtained by PFPP-MS/MS, suggesting this single approach has the capability of providing comprehensive metabolite profiling similar to the combination of methods. The more accurate quantification obtained by this method forms a fundamental basis for flux measurements and can be used for metabolism modeling in bacteria in future studies. PMID:20950815

  3. Quantification of peptide m/z distributions from 13C-labeled cultures with high resolution mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    With the introduction of orbital trap mass spectrometers molecular masses can be determined with great precision and accuracy. In addition, orbital trap spectrometers (Orbitraps) are sensitive and possess a linear dynamic range of multiple orders of magnitude. These qualities make the Orbitrap well-...

  4. Balancing the (carbon) budget: Using linear inverse models to estimate carbon flows and mass-balance 13C:15N labelling experiments in low oxygen sediments.

    NASA Astrophysics Data System (ADS)

    Hunter, William Ross; Van Oevelen, Dick; Witte, Ursula

    2013-04-01

    Over 1 million km2 of seafloor experience permanent low-oxygen conditions within oxygen minimum zones (OMZs). OMZs are predicted to grow as a consequence of climate change, potentially affecting oceanic biogeochemical cycles. The Arabian Sea OMZ impinges upon the western Indian continental margin at bathyal depths (150 - 1500m) producing a strong depth dependent oxygen gradient at the sea floor. The influence of the OMZ upon the short term processing of organic matter by sediment ecosystems was investigated using in situ stable isotope pulse chase experiments. These deployed doses of 13C:15N labeled organic matter onto the sediment surface at four stations from across the OMZ (water depth 540 - 1100 m; [O2] = 0.35 - 15 μM). In order to prevent experimentally anoxia, the mesocosms were not sealed. 13C and 15N labels were traced into sediment, bacteria, fauna and 13C into sediment porewater DIC and DOC. However, the DIC and DOC flux to the water column could not be measured, limiting our capacity to obtain mass-balance for C in each experimental mesocosm. Linear Inverse Modeling (LIM) provides a method to obtain a mass-balanced model of carbon flow that integrates stable-isotope tracer data with community biomass and biogeochemical flux data from a range of sources. Here we present an adaptation of the LIM methodology used to investigate how ecosystem structure influenced carbon flow across the Indian margin OMZ. We demonstrate how oxygen conditions affect food-web complexity, affecting the linkages between the bacteria, foraminifera and metazoan fauna, and their contributions to benthic respiration. The food-web models demonstrate how changes in ecosystem complexity are associated with oxygen availability across the OMZ and allow us to obtain a complete carbon budget for the stationa where stable-isotope labelling experiments were conducted.

  5. Probing the unfolding of myoglobin and domain C of PARP-1 with covalent labeling and top-down ultraviolet photodissociation mass spectrometry.

    PubMed

    Cammarata, Michael; Lin, Ke-Yi; Pruet, Jeff; Liu, Hung-Wen; Brodbelt, Jennifer

    2014-03-01

    Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins. PMID:24484264

  6. Determination of sup 13 C labeling pattern of citric acid cycle intermediates by gas chromatography-mass spectrometry

    SciTech Connect

    Di Donato, L.; Montgomery, J.A.; Des Rosiers, C.; David, F.; Garneau, M.; Brunengraber, H. )

    1990-02-26

    Investigations of the regulation of the citric acid cycle require determination of labeling patterns of cycle intermediates. These were assayed to date, using infusion of: (i) ({sup 14}C)tracer followed by chemical degradation of intermediates and (ii) ({sup 13}C)tracer followed by NMR analysis of intermediates. The authors developed a strategy to analyze by GC-MS the ({sup 13}C) labeling pattern of {mu}mole samples of citrate (CIT), isocitrate (ICIT), 2-ketoglutarate (2-KG), glutamate (GLU) and glutamine (GLN). These are enzymatically or chemically converted to 2-KG, ICIT, 4-aminobutyrate (GABA) and 2-hydroxyglutarate (2-OHG). GC-MS analyses of TMS or TBDMS derivatives of these compounds yield the enrichment of each carbon. The authors confirmed the identity of each fragment using the spectra of (1-{sup 13}C), (5-{sup 13}C), (2,3,3,4,4-{sup 2}H{sub 5})glutamate and (1-{sup 13}C), (1,4-{sup 13}C)GABA.

  7. Non-targeted determination of (13)C-labeling in the Methylobacterium extorquens AM1 metabolome using the two-dimensional mass cluster method and principal component analysis.

    PubMed

    Reaser, Brooke C; Yang, Song; Fitz, Brian D; Parsons, Brendon A; Lidstrom, Mary E; Synovec, Robert E

    2016-02-01

    A novel analytical workflow is presented for the analysis of time-dependent (13)C-labeling of the metabolites in the methylotrophic bacterium Methylobacterium extorquens AM1 using gas chromatography time-of-flight mass spectrometry (GC-TOFMS). Using (13)C-methanol as the substrate in a time course experiment, the method provides an accurate determination of the number of carbons converted to the stable isotope. The method also extracts a quantitative isotopic dilution time course profile for (13)C uptake of each metabolite labeled that could in principle be used to obtain metabolic flux rates. The analytical challenges encountered require novel analytical platforms and chemometric techniques. GC-TOFMS offers advanced separation of mixtures, identification of individual components, and high data density for the application of advanced chemometrics. This workflow combines both novel and traditional chemometric techniques, including the recently reported two-dimensional mass cluster plot method (2D m/z cluster plot method) as well as principal component analysis (PCA). The 2D m/z cluster plot method effectively indexed all metabolites present in the sample and deconvoluted metabolites at ultra-low chromatographic resolution (RS≈0.04). Using the pure mass spectra extracted, two PCA models were created. Firstly, PCA was used on the first and last time points of the time course experiment to determine and quantify the extent of (13)C uptake. Secondly, PCA modeled the full time course in order to quantitatively extract the time course profile for each metabolite. The 2D m/z cluster plot method found 152 analytes (metabolites and reagent peaks), with 54 pure analytes, and 98 were convoluted, with 65 of the 98 requiring mathematical deconvolution. Of the 152 analytes surveyed, 83 were metabolites determined by the PCA model to have incorporated (13)C while 69 were determined to be either metabolites or reagent peaks that remained unlabeled. PMID:26787164

  8. Interfacing droplet microfluidics with matrix-assisted laser desorption/ionization mass spectrometry: label-free content analysis of single droplets.

    PubMed

    Küster, Simon K; Fagerer, Stephan R; Verboket, Pascal E; Eyer, Klaus; Jefimovs, Konstantins; Zenobi, Renato; Dittrich, Petra S

    2013-02-01

    Droplet-based microfluidic systems have become a very powerful tool to miniaturize chemical and biological reactions. However, droplet content analysis remains challenging and relies almost exclusively on optical methods such as fluorescence spectroscopy. Hence, labeling of the analyte is typically required which impedes a more universal applicability of microdroplets. Here we present a novel interface coupling droplet microfluidics and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for label-free content analysis of single droplets. Nanoliter aqueous droplets immersed in perfluorinated oil are created in a microfluidic T-junction, transferred into a capillary, and deposited on a high-density microarray MALDI plate mounted on a motorized xy-stage. The fully automated system is robust and reliable due to two unique features. First, a simple optical droplet detection system is used to synchronize stage movement and exit of droplets from the capillary. Second, the microarray plate contains an array of over 26,000 hydrophilic spots within a hydrophobic coating, each spot acting as a recipient to confine the droplets and to prevent cross-contamination. The MALDI matrix can also be applied using our system by spotting matrix droplets on the microarray in a separate run. To demonstrate the potential of our system, we studied the enzymatic cleavage of angiotensin I by angiotensin converting enzyme and monitored the increasing concentration of the product angiotensin II over time. The interface provides a robust and fully automated method for rapid label-free and information-rich content analysis of single droplets. With the high number of droplets per plate, this method is particularly suitable for high-throughput screening applications. PMID:23289755

  9. Differential binding of ppGpp and pppGpp to E. coli RNA polymerase: photo-labeling and mass spectral studies.

    PubMed

    Syal, Kirtimaan; Chatterji, Dipankar

    2015-12-01

    (p)ppGpp, a secondary messenger, is induced under stress and shows pleiotropic response. It binds to RNA polymerase and regulates transcription in Escherichia coli. More than 25 years have passed since the first discovery was made on the direct interaction of ppGpp with E. coli RNA polymerase. Several lines of evidence suggest different modes of ppGpp binding to the enzyme. Earlier cross-linking experiments suggested that the β-subunit of RNA polymerase is the preferred site for ppGpp, whereas recent crystallographic studies pinpoint the interface of β'/ω-subunits as the site of action. With an aim to validate the binding domain and to follow whether tetra- and pentaphosphate guanosines have different location on RNA polymerase, this work was initiated. RNA polymerase was photo-labeled with 8-azido-ppGpp/8-azido-pppGpp, and the product was digested with trypsin and subjected to mass spectrometry analysis. We observed three new peptides in the trypsin digest of the RNA polymerase labeled with 8-azido-ppGpp, of which two peptides correspond to the same pocket on β'-subunit as predicted by X-ray structural analysis, whereas the third peptide was mapped on the β-subunit. In the case of 8-azido-pppGpp-labeled RNA polymerase, we have found only one cross-linked peptide from the β'-subunit. However, we were unable to identify any binding site of pppGpp on the β-subunit. Interestingly, we observed that pppGpp at high concentration competes out ppGpp bound to RNA polymerase more efficiently, whereas ppGpp cannot titrate out pppGpp. The competition between tetraphosphate guanosine and pentaphosphate guanosine for E. coli RNA polymerase was followed by gel-based assay as well as by a new method known as DRaCALA assay. PMID:26606426

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

    SciTech Connect

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

    2014-08-25

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

  11. Differentiation of the Stereochemistry and Anomeric Configuration for 1-3 Linked Disaccharides Via Tandem Mass Spectrometry and 18O-labeling

    NASA Astrophysics Data System (ADS)

    Konda, Chiharu; Bendiak, Brad; Xia, Yu

    2012-02-01

    Collision-induced dissociation (CID) of deprotonated hexose-containing disaccharides ( m/z 341) with 1-2, 1-4, and 1-6 linkages yields product ions at m/z 221, which have been identified as glycosyl-glycolaldehyde anions. From disaccharides with these linkages, CID of m/z 221 ions produces distinct fragmentation patterns that enable the stereochemistries and anomeric configurations of the non-reducing sugar units to be determined. However, only trace quantities of m/z 221 ions can be generated for 1-3 linkages in Paul or linear ion traps, preventing further CID analysis. Here we demonstrate that high intensities of m/z 221 ions can be built up in the linear ion trap (Q3) from beam-type CID of a series of 1-3 linked disaccharides conducted on a triple quadrupole/linear ion trap mass spectrometer. 18O-labeling at the carbonyl position of the reducing sugar allowed mass-discrimination of the "sidedness" of dissociation events to either side of the glycosidic linkage. Under relatively low energy beam-type CID and ion trap CID, an m/z 223 product ion containing 18O predominated. It was a structural isomer that fragmented quite differently than the glycosyl-glycolaldehydes and did not provide structural information about the non-reducing sugar. Under higher collision energy beam-type CID conditions, the formation of m/z 221 ions, which have the glycosyl-glycolaldehyde structures, were favored. Characteristic fragmentation patterns were observed for each m/z 221 ion from higher energy beam-type CID of 1-3 linked disaccharides and the stereochemistry of the non-reducing sugar, together with the anomeric configuration, were successfully identified both with and without 18O-labeling of the reducing sugar carbonyl group.

  12. Mass

    SciTech Connect

    Chris Quigg

    2007-12-05

    In the classical physics we inherited from Isaac Newton, mass does not arise, it simply is. The mass of a classical object is the sum of the masses of its parts. Albert Einstein showed that the mass of a body is a measure of its energy content, inviting us to consider the origins of mass. The protons we accelerate at Fermilab are prime examples of Einsteinian matter: nearly all of their mass arises from stored energy. Missing mass led to the discovery of the noble gases, and a new form of missing mass leads us to the notion of dark matter. Starting with a brief guided tour of the meanings of mass, the colloquium will explore the multiple origins of mass. We will see how far we have come toward understanding mass, and survey the issues that guide our research today.

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

    SciTech Connect

    Svensson, Jan; Bergman, Ann-Charlotte; Adamson, Ulf; Blombaeck, Margareta; Wallen, Hakan; Joerneskog, Gun

    2012-05-04

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

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

    PubMed

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

    2014-07-01

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

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

    PubMed

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

    2016-04-01

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

  16. Variation in airborne 137Cs peak levels with altitude from high-altitude locations across Europe after the arrival of Fukushima-labeled air masses

    NASA Astrophysics Data System (ADS)

    Masson, Olivier; Bieringer, Jacqueline; Dalheimer, Axel; Estier, Sybille; Evrard, Olivier; Penev, Ilia; Ringer, Wolfgang; Schlosser, Clemens; Steinkopff, Thomas; Tositti, Laura; de Vismes-Ott, Anne

    2015-04-01

    During the Fukushima Daiichi nuclear power plant (FDNPP) accident, a dozen of high-altitude aerosol sampling stations, located between 850 and 3,454 m above sea level (a.s.l.), provided airborne activity levels across Europe (Fig. 1). This represents at most 5% of the total number of aerosol sampling locations that delivered airborne activity levels (at least one result) in Europe, in connection with this nuclear accident. High altitude stations are typically equipped with a high volume sampler that collects aerosols on filters. The Fukushima-labeled air mass arrival and the peak of airborne cesium-137 (137Cs) activity levels were registered in Europe at different dates depending on the location, with differences up to a factor of six on a regional scale. Besides this statement related to lowland areas, we have compared the maximum airborne levels registered at high-altitude European locations (850 m < altitudes < 3450 m) with what was observed at the closest lowland location. The vertical distribution of 137Cs peak level was not uniform even after a long travel time/distance from Japan. This being true at least in the atmospheric boundary layer and in the lower free troposphere. Moreover the relation '137Csmax vs. altitude' shows a decreasing trend (Fig. 2). Results and discussion : Comparison of 137Cs and 7Be levels shows simultaneous increases at least when the 137Cs airborne level rose for the first time (Fig. 3). Zugspitze and Jungfraujoch stations attest of a time shift between 7Be and 137Cs peak that can be due to the particular dynamic of air movements at such high altitudes. After the 137Cs peak value, the plume concentration decreased whatever the 7Be level. Due to the cosmogenic origin of 7Be, its increase in the ground-level air is usually associated with downwind air movements, i.e. stratospheric air intrusions or at least air from high-tropospheric levels, into lower atmospheric layers. This means that Fukushima-labeled air masses registered at ground

  17. Measurement of rimantadine in plasma by capillary gas chromatography/mass spectrometry with a deuterium-labeled internal standard

    SciTech Connect

    Herold, D.A.; Anonick, P.K.; Kinter, M.; Hayden, F.G.

    1988-08-01

    Rimantadine is a synthetic antiviral agent used in prophylaxis and in treating the early stages of uncomplicated influenza A illness. We describe a stable isotope-dilution assay involving capillary gas chromatography/mass spectrometry. We used 200 ng of d3-rimantadine, added to 1 mL of plasma, as the internal standard. The rimantadine was extracted from the plasma with a Bond-Elut CN column, the column was washed with water, and the rimantadine was eluted with methanol, dried, and treated to form the t-butyldimethylsilyl derivative. The mass spectrometer was operated in the selected ion monitoring mode. Ions at m/z 236 and m/z 239 were monitored, corresponding to the loss of C4H9 from the rimantadine derivative and d3-rimantadine, respectively. Within-run precision (CVs) ranged from 8.9% at 29 micrograms/L to 3.2% at 1666 micrograms/L. Corresponding data for between-run precision were 5.4% and 1.7%. Treated volunteers (n = 86) provided plasma samples with a concentration range of 153 to 1127 micrograms/L. This simplified method allows rapid, precise assay of rimantadine in plasma.

  18. Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry.

    PubMed

    Wang, Chunlei; Chen, Sike; Brailsford, John A; Yamniuk, Aaron P; Tymiak, Adrienne A; Zhang, Yingru

    2015-12-24

    Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid. PMID:26674608

  19. Selective Analysis of Sulfur-Containing Species in a Heavy Crude Oil by Deuterium Labeling Reactions and Ultrahigh Resolution Mass Spectrometry

    PubMed Central

    Wang, Xuxiao; Schrader, Wolfgang

    2015-01-01

    A heavy crude oil has been treated with deuterated alkylating reagents (CD3I and C2D5I) and directly analyzed without any prior fractionation and chromatographic separation by high-field Orbitrap Fourier Transform Mass Spectrometry (FTMS) and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) using electrospray ionization (ESI). The reaction of a polycyclic aromatic sulfur heterocycles (PASHs) dibenzothiophene (DBT), in the presence of silver tetrafluoroborate (AgBF4) with ethyl iodide (C2H5I) in anhydrous dichloroethane (DCE) was optimized as a sample reaction to study heavy crude oil mixtures, and the reaction yield was monitored and determined by proton nuclear magnetic resonance spectroscopy (1H-NMR). The obtained conditions were then applied to a mixture of standard aromatic CH-, N-, O- and S-containing compounds and then a heavy crude oil, and only sulfur-containing compounds were selectively alkylated. The deuterium labeled alkylating reagents, iodomethane-d3 (CD3I) and iodoethane-d5 (C2D5I), were employed to the alkylation of heavy crude oil to selectively differentiate the tagged sulfur species from the original crude oil. PMID:26694374

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

    PubMed Central

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

    2010-01-01

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

  1. Sensitive electrospray mass spectrometry analysis of one-bead-one-compound peptide libraries labeled by quaternary ammonium salts.

    PubMed

    Bąchor, Remigiusz; Cydzik, Marzena; Rudowska, Magdalena; Kluczyk, Alicja; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2012-08-01

    A rapid and straightforward method for high-throughput analysis of single resin beads from one-bead-one-compound combinatorial libraries with high resolution electrospray ionization tandem mass spectrometry (HR ESI-MS/MS) is presented. The application of an efficient method of peptide derivatization by quaternary ammonium salts (QAS) formation increases ionization efficiency and reduces the detection limit, allowing analysis of trace amounts of compounds by ESI-MS. Peptides, synthesized on solid support, contain a new cleavable linker composed of a Peg spacer (9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid), lysine with ɛ-amino group marked by the N,N,N-triethylglycine salt, and methionine, which makes possible the selective cleavage by cyanogen bromide. Even a small portion of peptides derivatized by QAS cleaved from a single resin bead is sufficient for sequencing by HR ESI-MS/MS experiments. The developed strategy was applied to a small training library of α chymotrypsin substrates. The obtained results confirm the applicability of the proposed method in combinatorial chemistry. PMID:22740104

  2. 13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans

    PubMed Central

    Drechsler, Robin; Gafken, Philip R.; Olsen, Carissa Perez

    2015-01-01

    Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in C. elegans combining 13C and 15N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism. PMID:26528916

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

    PubMed

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

    2016-08-01

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

  4. CVD Prevention Through Policy: a Review of Mass Media, Food/Menu Labeling, Taxation/Subsidies, Built Environment, School Procurement, Worksite Wellness, and Marketing Standards to Improve Diet.

    PubMed

    Afshin, Ashkan; Penalvo, Jose; Del Gobbo, Liana; Kashaf, Michael; Micha, Renata; Morrish, Kurtis; Pearson-Stuttard, Jonathan; Rehm, Colin; Shangguan, Siyi; Smith, Jessica D; Mozaffarian, Dariush

    2015-11-01

    Poor diet is the leading cause of cardiovascular disease in the USA and globally. Evidence-based policies are crucial to improve diet and population health. We reviewed the effectiveness for a range of policy levers to alter diet and diet-related risk factors. We identified evidence to support benefits of focused mass media campaigns (especially for fruits, vegetables, salt), food pricing strategies (both subsidies and taxation, with stronger effects at lower income levels), school procurement policies (for increasing healthful or reducing unhealthful choices), and worksite wellness programs (especially when comprehensive and multicomponent). Evidence was inconclusive for food and menu labeling (for consumer or industry behavior) and changes in local built environment (e.g., availability or accessibility of supermarkets, fast food outlets). We found little empiric evidence evaluating marketing restrictions, although broad principles and large resources spent on marketing suggest utility. Widespread implementation and evaluation of evidence-based policy strategies, with further research on other strategies with mixed/limited evidence, are essential "population medicine" to reduce health and economic burdens and inequities of diet-related illness worldwide. PMID:26370554

  5. Review, evaluation, and discussion of the challenges of missing value imputation for mass spectrometry-based label-free global proteomics

    DOE PAGESBeta

    Webb-Robertson, Bobbie-Jo M.; Wiberg, Holli K.; Matzke, Melissa M.; Brown, Joseph N.; Wang, Jing; McDermott, Jason E.; Smith, Richard D.; Rodland, Karin D.; Metz, Thomas O.; Pounds, Joel G.; et al

    2015-04-09

    In this review, we apply selected imputation strategies to label-free liquid chromatography–mass spectrometry (LC–MS) proteomics datasets to evaluate the accuracy with respect to metrics of variance and classification. We evaluate several commonly used imputation approaches for individual merits and discuss the caveats of each approach with respect to the example LC–MS proteomics data. In general, local similarity-based approaches, such as the regularized expectation maximization and least-squares adaptive algorithms, yield the best overall performances with respect to metrics of accuracy and robustness. However, no single algorithm consistently outperforms the remaining approaches, and in some cases, performing classification without imputation sometimes yieldedmore » the most accurate classification. Thus, because of the complex mechanisms of missing data in proteomics, which also vary from peptide to protein, no individual method is a single solution for imputation. In summary, on the basis of the observations in this review, the goal for imputation in the field of computational proteomics should be to develop new approaches that work generically for this data type and new strategies to guide users in the selection of the best imputation for their dataset and analysis objectives.« less

  6. Review, evaluation, and discussion of the challenges of missing value imputation for mass spectrometry-based label-free global proteomics

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; Wiberg, Holli K.; Matzke, Melissa M.; Brown, Joseph N.; Wang, Jing; McDermott, Jason E.; Smith, Richard D.; Rodland, Karin D.; Metz, Thomas O.; Pounds, Joel G.; Waters, Katrina M.

    2015-04-09

    In this review, we apply selected imputation strategies to label-free liquid chromatography–mass spectrometry (LC–MS) proteomics datasets to evaluate the accuracy with respect to metrics of variance and classification. We evaluate several commonly used imputation approaches for individual merits and discuss the caveats of each approach with respect to the example LC–MS proteomics data. In general, local similarity-based approaches, such as the regularized expectation maximization and least-squares adaptive algorithms, yield the best overall performances with respect to metrics of accuracy and robustness. However, no single algorithm consistently outperforms the remaining approaches, and in some cases, performing classification without imputation sometimes yielded the most accurate classification. Thus, because of the complex mechanisms of missing data in proteomics, which also vary from peptide to protein, no individual method is a single solution for imputation. In summary, on the basis of the observations in this review, the goal for imputation in the field of computational proteomics should be to develop new approaches that work generically for this data type and new strategies to guide users in the selection of the best imputation for their dataset and analysis objectives.

  7. Quantitative analysis of 15N labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enteral metabolism of glutamine and citrulline are intertwined because, while glutamine is one of the main fuel sources for the enterocyte, citrulline is one of its products. It has been shown that the administration of 15N labeled glutamine results in the incorporation of the 15N label into cit...

  8. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

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

  9. Increased Depth and Breadth of Plasma Protein Quantitation via Two-Dimensional Liquid Chromatography/Multiple Reaction Monitoring-Mass Spectrometry with Labeled Peptide Standards.

    PubMed

    Percy, Andrew J; Yang, Juncong; Chambers, Andrew G; Borchers, Christoph H

    2016-01-01

    Absolute quantitative strategies are emerging as a powerful and preferable means of deriving concentrations in biological samples for systems biology applications. Method development is driven by the need to establish new-and validate current-protein biomarkers of high-to-low abundance for clinical utility. In this chapter, we describe a methodology involving two-dimensional (2D) reversed-phase liquid chromatography (RPLC), operated under alkaline and acidic pH conditions, combined with multiple reaction monitoring (MRM)-mass spectrometry (MS) (also called selected reaction monitoring (SRM)-MS) and a complex mixture of stable isotope-labeled standard (SIS) peptides, to quantify a broad and diverse panel of 253 proteins in human blood plasma. The quantitation range spans 8 orders of magnitude-from 15 mg/mL (for vitamin D-binding protein) to 450 pg/mL (for protein S100-B)-and includes 31 low-abundance proteins (defined as being <10 ng/mL) of potential disease relevance. The method is designed to assess candidates at the discovery and/or verification phases of the biomarker pipeline and can be adapted to examine smaller or alternate panels of proteins for higher sample throughput. Also detailed here is the application of our recently developed software tool-Qualis-SIS-for protein quantitation (via regression analysis of standard curves) and quality assessment of the resulting data. Overall, this chapter provides the blueprint for the replication of this quantitative proteomic method by proteomic scientists of all skill levels. PMID:26867735

  10. Specific deuterium labelling and computerized gas chromatography -- mass spectrometry in studies on the metabolism in vivo of a steroid sulphate in the rat.

    PubMed

    Baillie, T A; Eriksson, H; Herz, J E; Sjövall, J

    1975-06-16

    The metabolism of 3beta-hydroxy-5alpha-pregnan-20-one sulphate was studied in bile fistula rats and in isolated perfused livers. Computerized gas chromatography--mass spectrometry, in combination with specific deuterium-labelling, was employed to follow the metabolic transformations. Male animals excreted metabolites into bile more rapidly than females, a finding which could be correlated with the preferential formation of glucuronide conjugates in the male liver. The major metabolic pathway in male rats involved the steps: hydrolysis, 2alpha-hydroxylation, oxidoreduction at C-3 and glucuronide conjugation, yielding 2alpha, 3alpha-dihydroxy-5alpha-pregnan-20-one glucuronide as the major metabolite. Only traces of the injected steroid sulphate were detected in bile from male animals. In contrast, the administered compound was the major steroid excreted in bile of female rats, where the main metabolite was identified as 3beta,15beta-dihydroxy-5alpha-pregnan-20-one sulphate. A minor metabolite, 3beta,16alpha-dihydroxy-5alpha-pregnan-20-one, was found as a monosulphate in female rats and as both a disulphate and a glucuronide conjugate in male rats. The deuterium content of the sulphated 15beta-and 16alpha-hydroxylated metabolites was consistent with metabolic pathways involving direct hydroxylation of the injected steroid sulphate. The results obtained from the liver perfusions were essentially the same as those from the experiments with bile fistula animals. This indicates that all the observed metabolic reactions took place in the liver. PMID:1175601

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

    PubMed

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

    2016-05-01

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

  12. Label scrambling during CID of covalently labeled peptide ions.

    PubMed

    Borotto, Nicholas B; Degraan-Weber, Nicholas; Zhou, Yuping; Vachet, Richard W

    2014-10-01

    Covalent labeling along with mass spectrometry is finding more use as a means of studying the higher order structure of proteins and protein complexes. Diethylpyrocarbonate (DEPC) is an increasingly used reagent for these labeling experiments because it is capable of modifying multiple residues at the same time. Pinpointing DEPC-labeled sites on proteins is typically needed to obtain more resolved structural information, and tandem mass spectrometry after protein proteolysis is often used for this purpose. In this work, we demonstrate that in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low charge-state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification. PMID:25056863

  13. Boronic acid recognition based-gold nanoparticle-labeling strategy for the assay of sialic acid expression on cancer cell surface by inductively coupled plasma mass spectrometry.

    PubMed

    Zhang, Xing; Chen, Beibei; He, Man; Zhang, Yuan; Peng, Lu; Hu, Bin

    2016-02-01

    Sialic acids are special sugars widely expressed at the termini of glycan chains on the cell surface, and their expression level on the cancer cell surface is much higher than on the normal cell surface. Herein, we reported an inductively coupled plasma mass spectrometry (ICP-MS) based method with elemental tags for the analysis of sialic acids on the cancer cell surface. The method is based on the selective recognition of sialic acids by biotinylated phenylboronic acid (biotin-APBA) at physiological pH and signal enhancement of gold nanoparticles (AuNPs) in ICP-MS when AuNPs were used as elemental tags labeled on biotin-APBA. A specificity test reveals that the proposed method has high specificity towards cancer cells. Taking HepG2 and MCF-7 cells as two model cancer cells, competitive experiments were performed to estimate the expression level of sialic acids on the cancer cell surface, and it was found that the average numbers of sialic acids expressed on the single MCF-7 and HepG2 cell surface were 7.0 × 10(9) and 5.4 × 10(9), respectively. With sialic acid as the biomarker for cancer cells, the method was further used for cell detection. The limits of detection in terms of cell number for HepG2 and MCF-7 cells were 120 and 64, respectively. And the relative standard deviations for nine replicate determinations of ca. 1000 HepG2 and MCF-7 cells were 9.6% and 8.9%, respectively. The linear ranges for HepG2 cells and MCF-7 cells were 300-10 000 and 170-11 000, respectively. The proposed approach is sensitive as well as selective for the analysis of sialic acids on the cancer cell surface, and is potentially applicable for the study of tumor malignancy and metastasis, which is helpful for biological research and clinical diagnostics. PMID:26811850

  14. Transformation of 17β-estradiol in humic acid solution by ε-MnO2 nanorods as probed by high-resolution mass spectrometry combined with (13)C labeling.

    PubMed

    Sun, Kai; Liang, Shangtao; Kang, Fuxing; Gao, Yanzheng; Huang, Qingguo

    2016-07-01

    Steroidal estrogens (SEs), widespread in aquatic systems, have a potential to disrupt the endocrine system of wildlife species and humans. In our experiments, the performance of ε-MnO2 nanorods in transforming 17β-estradiol (E2) was investigated, and the effect of humic acid (HA) on the reaction behaviors was systematically characterized. Reconfiguration of humic molecules was also investigated by high-performance size exclusion chromatography (HPSEC). Results indicated that ε-MnO2 nanomaterials ensured efficient removal of E2 from the aqueous solution. The presence of HA hindered the transformation of E2, while enhanced the cross-coupling between E2 and humic molecules. In particular, we used a mixture of un-labeled E2 and (13)C3-labeled E2 at a 1: 1 set ratio (w/w) to probe the reaction products via high-resolution mass spectrometry (HRMS). The combination of HRMS and (13)C3-labeling revealed the intermediate products including estrone (E1), and hydroxylated, quinone-like, and ring-opened species, as well as E2 dimer and trimer. More importantly, possible cross-coupling products between E2 and HA were also identified. A reaction mechanism including two-electron oxidation and single-electron oxidation was proposed. The applied analytical approach using HRMS along with (13)C3-labeling for reaction-product identification is crucial to understanding the role of HA in the transformation of SEs. PMID:27086077

  15. μPET imaging of the pharmacokinetic behavior of medium and high molar mass (89)Zr-labeled poly(2-ethyl-2-oxazoline) in comparison to poly(ethylene glycol).

    PubMed

    Wyffels, Leonie; Verbrugghen, Thomas; Monnery, Bryn D; Glassner, Mathias; Stroobants, Sigrid; Hoogenboom, Richard; Staelens, Steven

    2016-08-10

    Poly(2-oxazoline)s are a promising class of polymers for biomedical applications and a versatile alternative to poly(ethylene glycol)s (PEG). In this work, the pharmacokinetic behavior of well defined (89)Zr-labeled poly(2-ethyl-2-oxazoline)s (PEtOx) was evaluated and compared to that of (89)Zr-labeled PEG, both with varying molar mass. Amine-terminated PEtOx of low dispersity in a molar mass range of 20 to 110kDa and PEG of 20 and 40kDa were functionalized with a desferrioxamine chelator and radiolabeled with (89)Zr. The tissue distribution of both radiolabeled PEtOx and PEG polymers was studied by means of micro Positron Emission Tomography (μPET) molecular imaging in mice longitudinally up to 1week post injection, followed by ex vivo biodistribution. As previously described for other classes of non-ionic polymers, the blood clearance of PEtOx decreased with molar mass. The cut off for glomerular filtration of PEtOx is likely to be around 40kDa. The head to head comparison of PEG and PEtOx revealed that the biodistribution is mostly dominated by polymer chain length and not polymer molar mass. This study constitutes an important addition to further establishing PEtOx as a promising polymer in biomedical applications. PMID:27235979

  16. Atmospheric pressure gas chromatography coupled to quadrupole-time of flight mass spectrometry as a tool for identification of volatile migrants from autoadhesive labels used for direct food contact.

    PubMed

    Canellas, Elena; Vera, Paula; Nerín, Cristina

    2014-11-01

    Pressure-sensitive adhesives (PSA) are used to manufacture labels that are applied directly on the food. These adhesives could contain not only intentionally added compounds (IAS) to the adhesive formula but also non-intentionally added substances (NIAS), due to the impurities from the raw materials used, decomposition of the initial components or from chemical interactions between them. These compounds could migrate to the food and contaminate it. In this study, gas chromatography coupled with mass spectrometry (GC-MS/Q) and atmospheric pressure gas chromatography coupled to a quadrupole hyphenated to a time of flight mass spectrometer (APGC-MS/Q-TOF) have been used for identification of unknown compounds and NIAS coming from a PSA. Seven compounds were identified by GC-MS/Q, and other eight compounds remained initially unknown. The structure of these eight new compounds was elucidated by working with the spectra obtained by APGC-MS/Q-TOF. Finally, two different migration studies were carried out. The first one with Tenax as solid food simulant in contact with the paper label containing the adhesive and the second one with isooctane filled in a natural pork intestine where the label containing the adhesive was applied on the external side. The results are shown and discussed. PMID:25395134

  17. Studies on the biodegradation of fosfomycin: synthesis of 13C-labeled intermediates, feeding experiments with Rhizobium huakuii PMY1, and isolation of labeled amino acids from cell mass by HPLC.

    PubMed

    McGrath, John W; Hammerschmidt, Friedrich; Kählig, Hanspeter; Wuggenig, Frank; Lamprecht, Günther; Quinn, John P

    2011-11-18

    Racemic (1R*,2R*)-1,2-dihydroxy-[1-(13)C(1)]propylphosphonic acid and 1-hydroxy-[1-(13)C(1)]acetone were synthesized and fed to R. huakuii PMY1. Alanine and a mixture of valine and methionine were isolated as their N-acetyl derivatives from the cell hydrolysate by reversed-phase HPLC and analyzed by NMR spectroscopy. It was found that the carbon atoms of the respective carboxyl groups were highly (13)C-labeled (up to 65 %). Hydroxyacetone is therefore considered an obligatory intermediate of the biodegradation of fosfomycin by R. huakuii PMY1. PMID:22012897

  18. Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2(+) breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry.

    PubMed

    Calderón-González, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazán-Méndez, César Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Marí aEsther; Sánchez Del Pino, Manuel M; Gallegos-Pérez, José Luis; González-Márquez, Humberto; Hernández-Hernández, Jose Manuel; León-Ávila, Gloria; Rodríguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

    2015-09-01

    Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article "Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry" (Calderón-González et al. [1] in press). PMID:26217805

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

    PubMed

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

    1996-01-01

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

  20. Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: Sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrometry

    SciTech Connect

    Brinegar, A.C.; Cooper, G.; Stevens, A.; Hauer, C.R.; Shabanowitz, J.; Hunt, D.F.; Fox, J.E. )

    1988-08-01

    A wheat embryo cytokinin-binding protein was covalently modified with the radiolabeled photoaffinity ligand 2-azido-N{sup 6}-({sup 14}C)benzyladenine. A single labeled peptide was obtained after proteolytic digestion and isolation by reversed-phase and anion-exchange HPLC. Sequencing by classical Edman degradation identified 11 of the 12 residues but failed to identify the labeled amino acid. Analysis by laser photodissociation Fourier-transform mass spectrometry of 10 pmol of the peptide independently confirmed the Edman data and also demonstrated that the histidine residue nearest the C terminus (underlined) was modified by the reagent in the sequence Ala-Phe-Leu-Gln-Pro-Ser-His-His{und His}-Asp-Ala-Asp-Glu.

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

    PubMed

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

    1991-08-01

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

  2. Identification of the two essential groups in the family 3 beta-glucosidase from Flavobacterium meningosepticum by labelling and tandem mass spectrometric analysis.

    PubMed Central

    Chir, Jiunly; Withers, Stephen; Wan, Chin-Feng; Li, Yaw-Kuen

    2002-01-01

    beta-Glucosidase from Flavobacterium meningosepticum (Fbgl) catalyses the hydrolysis of beta-1,4-glucosidic bonds via a two-step double-displacement mechanism in which two amino acid residues act as nucleophile and acid/base catalyst. Definitive identification of these two residues is provided by the two active-site-directed inactivators, 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-d-glucoside (2FDNPG) and N-bromoacetyl-beta-d-glucosylamine (NBGN), which stoichiometrically label the nucleophile and the acid/base catalyst of Fbgl, respectively. Pseudo-first-order inactivation rate constants (k(i)) of 0.25+/-0.01 and 0.05+/-0.01 min(-1) and dissociation constants (K(i)) of 90+/-15 and 4.4+/-0.2 mM are determined for 2FDNPG and NBGN, respectively. Proteolytic digestion of the labelled proteins, followed by peptide mapping and tandem MS analysis identify Asp-247 and Glu-473 as the catalytic nucleophile and acid/base residues, respectively, of Fbgl. This study confirms that the catalytic nucleophile of family 3 glycohydrolase is conserved across sub-families. However, different sub-families may have unique general acid/base catalysts. PMID:11978178

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

    PubMed

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

    2015-04-01

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

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

    PubMed Central

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

    2015-01-01

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

  5. Spatial and temporal distribution of 13C labelled plant residues in soil aggregates and Lumbricus terrestris surface casts: A combination of Transmission Electron Microscopy and Nanoscale Secondary Ion Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Vidal, Alix; Remusat, Laurent; Watteau, Françoise; Derenne, Sylvie; Quenea, Katell

    2016-04-01

    Earthworms play a central role in litter decomposition, soil structuration and carbon cycling. They ingest both organic and mineral compounds which are mixed, complexed with mucus and dejected in form of casts at the soil surface and along burrows. Bulk isotopic or biochemical technics have often been used to study the incorporation of litter in soil and casts, but they could not reflect the complex interaction between soil, plant and microorganisms at the microscale. However, the heterogeneous distribution of organic carbon in soil structures induces contrasted microbial activity areas. Nano-scale secondary ion mass spectrometry (NanoSIMS), which is a high spatial resolution method providing elemental and isotopic maps of organic and mineral materials, has recently been applied in soil science (Herrmann et al., 2007; Vogel et al., 2014). The combination of Nano-scale secondary ion mass spectrometry (NanoSIMS) and Transmission Electron Microscopy (TEM) has proven its potential to investigate labelled residues incorporation in earthworm casts (Vidal et al., 2016). In line of this work, we studied the spatial and temporal distribution of plant residues in soil aggregates and earthworm surface casts. This study aimed to (1) identify the decomposition states of labelled plant residues incorporated at different time steps, in casts and soil, (2) identify the microorganisms implied in this decomposition (3) relate the organic matter states of decomposition with their 13C signature. A one year mesocosm experiment was set up to follow the incorporation of 13C labelled Ryegrass (Lolium multiflorum) litter in a soil in the presence of anecic earthworms (Lumbricus terrestris). Soil and surface cast samples were collected after 8 and 54 weeks, embedded in epoxy resin and cut into ultra-thin sections. Soil was fractionated and all and analyzed with TEM and NanoSIMS, obtaining secondary ion images of 12C, 16O, 12C14N, 13C14N and 28Si. The δ13C maps were obtained using the 13C14

  6. 4-Phenylaminomethyl-Benzeneboric Acid Modified Tip Extraction for Determination of Brassinosteroids in Plant Tissues by Stable Isotope Labeling-Liquid Chromatography-Mass Spectrometry.

    PubMed

    Yu, Lei; Ding, Jun; Wang, Ya-Lan; Liu, Ping; Feng, Yu-Qi

    2016-01-19

    Monitoring brassinosteroids (BRs) has been of major interest of researchers as these substances play a crucial role in a variety of phytological processes in plants. However, the determination of endogenous BRs in plant tissue is still a challenging task due to their low abundance and the complex matrix of plant tissues. In this study, a single step strategy by combining tip extraction and in situ derivatization was proposed for BR analysis. In the proposed strategy, a mixed mode sorbent (C8-SO3H) in tip was modified with 4-phenylaminomethyl-benzeneboric acid (4-PAMBA) through cation exchange and hydrophobic interactions, and then used as a boronate affinity media to selectively capture and purify BRs from plant extract through the reaction of boric acid groups of 4-PAMBA and cis-diol on BRs. The BRs-4-PAMBA derivatives formed were easily eluted from the C8-SO3H tip by nullifying the ion exchange and hydrophobic interactions using ammonia acetonitrile, followed by LC-MS/MS analysis. BR standards, isotopically labeled with d5-4-phenylaminomethyl-benzeneboric acid (4-PAMBA-d5) were introduced to improve the assay precision of LC-MS/MS. Under the optimized conditions, the overall process could be completed within 1 h, which is greatly improved in speed compared with previously reported protocols. In addition, the detection sensitivities of labeled BRs were improved by over 2000-fold compared with unlabeled BRs, thus the consumption of plant materials was reduced to 50 mg. Finally, the proposed method was applied for the investigation of BRs response in rice toward Cd stress. PMID:26650986

  7. Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2+ breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry

    PubMed Central

    Calderón-González, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazán-Méndez, César Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Marí;aEsther; Sánchez del Pino, Manuel M.; Gallegos-Pérez, José Luis; González-Márquez, Humberto; Hernández-Hernández, Jose Manuel; León-Ávila, Gloria; Rodríguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

    2015-01-01

    Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article “Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry” (Calderón-González et al. [1] in press). PMID:26217805

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

    PubMed

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

    2015-05-01

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

  9. abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes

    PubMed Central

    2014-01-01

    Affinity purification coupled to 1-D gel-free liquid chromatography mass spectrometry (LC–MS) is a well-established and widespread approach for the analyses of noncovalently interacting protein complexes. In this study, two proteins conjugated to a streptavidin-binding peptide and hemagglutinin double tag were expressed in the respective Flp-In HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed that the expression level of SH-GFP was ∼50% lower than that of SH-TBK1_MOUSE. Subsequently, the input material was normalized to obtain a similar quantity of purified SH-tagged proteins. Optimization of the release of protein complexes from the anti-HA-agarose with different eluting agents was then assessed. With respect to the total number of protein groups identified in the purified complexes, elution with 2% SDS surpassed both 100 mM glycine and 100 mM formic acid. Relative quantitation of the purified protein complexes using TMT 6-plex reagents confirmed the higher efficiency of the 2% SDS elution followed by filter-aided sample preparation (FASP). The data presented in this study provide a new application of FASP to quantitative MS analysis of affinity-purified protein complexes. We have termed the approach abFASP-MS, or affinity-based filter-aided sample preparation mass spectrometry. PMID:24400740

  10. Simultaneous measurements of endogenous and deuterium-labelled tracer variants of androstenedione and testosterone by capillary gas chromatography-mass spectrometry.

    PubMed

    Furuta, T; Kusano, K; Kasuya, Y

    1990-01-26

    A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of androstenedione and testosterone in human plasma using [19,19,19-2H3]androstenedione and [19,19,19-2H3]testosterone as internal standards is described. For calculation of plasma androstenedione and testosterone, peak heights were measured by selected-ion monitoring of the molecular ions of the heptafluorobutyryl derivatives of androstenedione and [2H3]androstenedione (m/z 482 and 485) and of testosterone and [2H3]testosterone (m/z 680 and 683). The isotope dilution method needed no complex corrections for contributions and provides a sensitive and reliable technique with good accuracy, precision and reproducibility. PMID:2338435

  11. Mass spectrometry.

    NASA Technical Reports Server (NTRS)

    Burlingame, A. L.; Johanson, G. A.

    1972-01-01

    Review of the current state of mass spectrometry, indicating its unique importance for advanced scientific research. Mass spectrometry applications in computer techniques, gas chromatography, ion cyclotron resonance, molecular fragmentation and ionization, and isotope labeling are covered. Details are given on mass spectrometry applications in bio-organic chemistry and biomedical research. As the subjects of these applications are indicated alkaloids, carbohydrates, lipids, terpenes, quinones, nucleic acid components, peptides, antibiotics, and human and animal metabolisms. Particular attention is given to the mass spectra of organo-inorganic compounds, inorganic mass spectrometry, surface phenomena such as secondary ion and electron emission, and elemental and isotope analysis. Further topics include mass spectrometry in organic geochemistry, applications in geochronology and cosmochemistry, and organic mass spectrometry.

  12. Application of Cassette Ultracentrifugation Using Non-labeled Compounds and Liquid Chromatography-Tandem Mass Spectrometry Analysis for High-Throughput Protein Binding Determination.

    PubMed

    Kieltyka, Kasia; McAuliffe, Brian; Cianci, Christopher; Drexler, Dieter M; Shou, Wilson; Zhang, Jun

    2016-03-01

    Membrane-based devices typically used for serum protein binding determination are not fully applicable to highly lipophilic compounds because of nonspecific binding to the device membrane. Ultracentrifugation, however, completely eliminates the issue by using a membrane-free approach, although its wide application has been limited. This lack of utilization is mainly attributed to 2 factors: the high cost in acquiring and handling of radiolabeled compounds and low assay throughput owing to the difficulties in process automation. To overcome these challenges, we report a high-throughput workflow by cassette ultracentrifugation of nonradiolabeled compounds followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Twenty compounds with diverse physicochemical and protein binding properties were selected for the evaluation of the workflow. To streamline the working process, approaches of matrix balancing for all the samples for LC-MS/MS analysis and determining free fraction without analytical calibration curves were adopted. Both the discrete ultracentrifugation of individual compounds and cassette ultracentrifugation of all the test compounds followed by simultaneous LC-MS/MS analysis exhibited a linear correlation with literature values, demonstrating respectively the validity of the ultracentrifugation process and the cassette approach. The cassette ultracentrifugation using nonradiolabeled compounds followed by LC-MS/MS analysis has greatly facilitated its application for high-throughput protein binding screening in drug discovery. PMID:26886323

  13. Unsupervised Identification of Isotope-Labeled Peptides.

    PubMed

    Goldford, Joshua E; Libourel, Igor G L

    2016-06-01

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

  14. Mass spectrometry-based microassay of (2)H and (13)C plasma glucose labeling to quantify liver metabolic fluxes in vivo.

    PubMed

    Hasenour, Clinton M; Wall, Martha L; Ridley, D Emerson; Hughey, Curtis C; James, Freyja D; Wasserman, David H; Young, Jamey D

    2015-07-15

    Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations. PMID:25991647

  15. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

    PubMed

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

    2016-08-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. PMID:27317400

  16. A novel quantification method for analysis of twenty natural amino acids in human serum based on N-phosphorylation labeling using reversed-phase liquid chromatography-tandem mass spectrometry.

    PubMed

    Chen, Xiaowu; Gao, Dan; Liu, Feng; Gao, Xiang; Wang, Shujuan; Zhao, Yufen; Liu, Hongxia; Jiang, Yuyang

    2014-07-11

    A novel method based on the strategy of N-phosphorylation labeling is described for quantification of twenty natural amino acids in human serum by reversed-phase liquid chromatography-electrospray tandem mass spectrometry (RP-LC/ESI-MS). The derivatization reaction was easily performed in one-pot reaction under mild conditions within 30min. The reaction mixture was then evaporated to dryness, redissolved, desalted by C18 SPE. The twenty N-phosphoryl amino acids were separated on an RP-C18 column within 20min by isocratic elution (0.1% formic acid-acetonitrile, v/v 7:3). At the same time, multiple reaction monitoring (MRM) MS enabled quantitation of twenty natural amino with the LOD of 0.0005-0.15μM and LOQ of 0.0020-0.5μM in human serum. The linear range was from 0.025 to 25μM (except Cys and Trp) with R>0.99. The recovery range was determined to be 85.5-117.4% with the relative standard deviation (RSD) in the range of 1.3-13.9%. All twenty amino acids were successfully detected in human serum samples with the concentration from 5.7 to 577.9μM, which indicates potential of the developed method for determination of amino acids in complex biological samples, hence for screening of amino acid metabolite related diseases. PMID:24974871

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

    PubMed

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

    2016-05-01

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

  18. Semiotic labelled deductive systems

    SciTech Connect

    Nossum, R.T.

    1996-12-31

    We review the class of Semiotic Models put forward by Pospelov, as well as the Labelled Deductive Systems developed by Gabbay, and construct an embedding of Semiotic Models into Labelled Deductive Systems.

  19. Mental Labels and Tattoos

    ERIC Educational Resources Information Center

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  20. Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

    SciTech Connect

    Li, Zhou; Adams, Rachel M; Chourey, Karuna; Hurst, Gregory {Greg} B; Hettich, Robert {Bob} L; Pan, Chongle

    2012-01-01

    A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaric chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.

  1. Trypsin-catalyzed oxygen-18 labeling for quantitative proteomics

    SciTech Connect

    Qian, Weijun; Petritis, Brianne O.; Nicora, Carrie D.; Smith, Richard D.

    2011-07-01

    Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In (18)O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with (18)O, thus providing the labeled peptide with a 4 Da mass shift from the (16)O-labeled sample. Peptide (18)O labeling is ideally suited for generating a labeled "universal" reference sample used for obtaining accurate and reproducible quantitative measurements across large number of samples in quantitative discovery proteomics.

  2. Bar Code Labels

    NASA Technical Reports Server (NTRS)

    1988-01-01

    American Bar Codes, Inc. developed special bar code labels for inventory control of space shuttle parts and other space system components. ABC labels are made in a company-developed anodizing aluminum process and consecutively marketed with bar code symbology and human readable numbers. They offer extreme abrasion resistance and indefinite resistance to ultraviolet radiation, capable of withstanding 700 degree temperatures without deterioration and up to 1400 degrees with special designs. They offer high resistance to salt spray, cleaning fluids and mild acids. ABC is now producing these bar code labels commercially or industrial customers who also need labels to resist harsh environments.

  3. Labeling and Delinquency.

    ERIC Educational Resources Information Center

    Adams, Mike S.; Robertson, Craig T.; Gray-Ray, Phyllis; Ray, Melvin C.

    2003-01-01

    Index comprised of six contrasting descriptive adjectives was used to measure incarcerated youths' perceived negative labeling from the perspective of parents, teachers, and peers. Results provided partial support for hypothesis that juveniles who choose a greater number of negative labels will report more frequent delinquent involvement. Labeling…

  4. Label fusion strategy selection.

    PubMed

    Robitaille, Nicolas; Duchesne, Simon

    2012-01-01

    Label fusion is used in medical image segmentation to combine several different labels of the same entity into a single discrete label, potentially more accurate, with respect to the exact, sought segmentation, than the best input element. Using simulated data, we compared three existing label fusion techniques-STAPLE, Voting, and Shape-Based Averaging (SBA)-and observed that none could be considered superior depending on the dissimilarity between the input elements. We thus developed an empirical, hybrid technique called SVS, which selects the most appropriate technique to apply based on this dissimilarity. We evaluated the label fusion strategies on two- and three-dimensional simulated data and showed that SVS is superior to any of the three existing methods examined. On real data, we used SVS to perform fusions of 10 segmentations of the hippocampus and amygdala in 78 subjects from the ICBM dataset. SVS selected SBA in almost all cases, which was the most appropriate method overall. PMID:22518113

  5. OR Specimen Labeling.

    PubMed

    Zervakis Brent, Mary Ann

    2016-02-01

    Mislabeled surgical specimens jeopardize patient safety and quality care. The purpose of this project was to determine whether labeling surgical specimens with two patient identifiers would result in an 80% reduction in specimen labeling errors within six months and a 100% reduction in errors within 12 months. Our failure mode effects analysis found that the lack of two patient identifiers per label was the most unsafe step in our specimen handling process. We piloted and implemented a new process in the OR using the Plan-Do-Check-Act conceptual framework. The audit process included collecting data and making direct observations to determine the sustainability of the process change; however, the leadership team halted the direct observation audit after four months. The total number of surgical specimen labeling errors was reduced by only 60% within six months and 62% within 12 months; therefore, the goal of the project was not met. However, OR specimen labeling errors were reduced. PMID:26849982

  6. Nanovehicles based Bioassay Labels

    SciTech Connect

    Liu, Guodong; Wang, Jun; Wu, Hong; Lin, Ying-Ying; Lin, Yuehe

    2007-04-01

    In this article, we review recent advances of our group in nanoparticle labels based bioassay. Apoferritin and silica nanoparticles have been used as nanovehicles to load large amount of markers for highly sensitive bioassay. Markers loaded apoferritin, apoferritin-templated metallic phosphate nanoparticles, and poly [guanine] coated silica nanoparticles have been prepared, characterized and used as labels for highly sensitive bioassay of protein and DNA. Dissociation and reconstitution characteristics at different pH as well as the special cavity structure of apoferritin nanovehicle provides a simple and convenient route to prepare versatile nanoparticle labels and avoid the complicated and tedious synthesis process of conventional nanoparticle labels. The optical and electrochemical characteristics of the prepared nanoparticle labels are easily controlled by loading different optical or electrochemical markers. Additionally, the use of apoferritin nanovehicle as template for synthesis of metallic phosphate nanoparticle labels offers fast route to prepare uniform-size metallic nanoparticle labels for electrochemical bioassay and avoids the traditional harsh dissolution conditions to dissolve metallic nanoparticle tags (that is, the strong-acid dissolution of quantum dots and gold nanoparticles) during the stripping analysis step. Silica nanoparticle has also been used as nanovehicle to carry thousands of poly [guanine] tracers, which was used to enhance the oxidation current of Ru(bpy)32+, resulting in enhanced sensitivity of electrochemical immunoassay. The new nanovehicle-based labels have been used for highly sensitive electrochemical detection of DNA and protein biomarkers, such as tumor necrosis factor-alpha (TNF-a). The high sensitivity and selectivity make these labels a useful addition to the armory of nanoparticle-based bioassay. The new nanovehicles based labels hold great promise for multiplex protein and DNA detection and for enhancing the sensitivity

  7. How to Read Drug Labels

    MedlinePlus

    ... and alternative medicine Healthy Aging How to read drug labels Printer-friendly version How to Read Drug ... read drug labels How to read a prescription drug label View a text version of this picture. ...

  8. Nutrition Facts: Reading the Label

    MedlinePlus

    ... My Go4Life Get Free Stuff Be a Partner Nutrition Facts: Reading the Label Reading labels can help ... of information on their labels or packaging about nutrition and food safety. Product dates . You might see ...

  9. Noise labeling in Brazil

    NASA Astrophysics Data System (ADS)

    de Araujo, Marco A. N.; Massarani, Paulo M.; de Azevedo, Jose A. J.; Gerges, Samir N. Y.

    2002-11-01

    The Brazilian Silence Program, created in 1990 by the Brazilian Ministry of Environment, advocates the production and use of equipment with lower noise level. The subcommittee of Noise Labeling of the Brazilian Committee of Certification is composed of INMETRO acoustic specialists to organize and implement the Brazilian Labeling Program. This subcommittee elaborated the label form and test procedure. The noise-labeling program will first concentrate on the following household devices, both manufactured in Brazil or imported from abroad; mixers, blenders, hairdryers, refrigerators, and vacuum cleaners. The label should contain the sound-power level in dBA. INMETRO or other credited laboratories are responsible for the measurements. The ISO 4871, 3740 (1 to 5), ISO 8960, and IEC 704 (1 to 4) and also the equivalent Brazilian standards are used for the measurements, such as ABNT NBR 13910-1. The main objective of the label is to inform the consumer about the emitted noise level. The label offers the noise parameter to be used by the consumer when comparing devices, considering price, performance, and now also noise. No restriction for noise level was established.

  10. High Resolution Quantitative Proteomics of HeLa Cells Protein Species Using Stable Isotope Labeling with Amino Acids in Cell Culture(SILAC), Two-Dimensional Gel Electrophoresis(2DE) and Nano-Liquid Chromatograpohy Coupled to an LTQ-OrbitrapMass Spectrometer*

    PubMed Central

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

    2013-01-01

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

  11. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1983-07-15

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  12. Capacitive label reader

    DOEpatents

    Arlowe, H. Duane

    1985-01-01

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  13. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1985-11-12

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label. 5 figs.

  14. Like your labels?

    PubMed

    Field, Michele

    2010-01-01

    The descriptive “conventions” used on food labels are always evolving. Today, however, the changes are so complicated (partly driven by legislation requiring disclosures about environmental impacts, health issues, and geographical provenance) that these labels more often baffle buyers than enlighten them. In a light-handed manner, the article points to how sometimes reading label language can be like deciphering runes—and how if we are familiar with the technical terms, we can find a literal meaning, but still not see the implications. The article could be ten times longer because food labels vary according to cultures—but all food-exporting cultures now take advantage of our short attention-span when faced with these texts. The question is whether less is more—and if so, in this contest for our attention, what “contestant” is voted off. PMID:21539053

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

    PubMed

    Price, John C; Ghaemmaghami, Sina

    2014-01-01

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

  16. Off-Label Drug Use

    MedlinePlus

    ... Your Local Offices Close + - Text Size Off-label Drug Use What is off-label drug use? In the United States new drugs are ... unapproved use of a drug. Is off-label drug use legal? The off-label use of FDA- ...

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

    PubMed

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

    2014-10-29

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

  18. Spectral Label Fusion

    PubMed Central

    Wachinger, Christian; Golland, Polina

    2012-01-01

    We present a new segmentation approach that combines the strengths of label fusion and spectral clustering. The result is an atlas-based segmentation method guided by contour and texture cues in the test image. This offers advantages for datasets with high variability, making the segmentation less prone to registration errors. We achieve the integration by letting the weights of the graph Laplacian depend on image data, as well as atlas-based label priors. The extracted contours are converted to regions, arranged in a hierarchy depending on the strength of the separating boundary. Finally, we construct the segmentation by a region-wise, instead of voxel-wise, voting, increasing the robustness. Our experiments on cardiac MRI show a clear improvement over majority voting and intensity-weighted label fusion. PMID:23286157

  19. Spin labeling EPR.

    PubMed

    Klare, Johann P; Steinhoff, Heinz-Jürgen

    2009-01-01

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

  20. Labeling the Children.

    ERIC Educational Resources Information Center

    Krasner, William

    The report describes research on the effects of labeling children from minority groups as retarded and includes a review of a system of multiculturalistic pluralistic assessment (SOMPA), an instrument for evaluating the abilities and potentialities of children based on different aspects of performance. Listed among findings of the Riverside study,…

  1. A Deceiving Label?

    ERIC Educational Resources Information Center

    Lum, Lydia

    2009-01-01

    The author reports on the growing debate among educators on whether the umbrella Asian Pacific Islander label conceals disparities among Asian American students or provides political power in numbers. Nationally, experts say that support services aimed at not only Southeast Asians, but all Asian Pacific Islander students, remain scarce in higher…

  2. Fluorous photoaffinity labeling to probe protein-small molecule interactions.

    PubMed

    Huang, Weigang; Zhang, Qisheng

    2015-01-01

    Identifying cellular targets of bioactive small molecules is essential for their applications as chemical probes or drug candidates. Of equal importance is to determine their "off-target" interactions, which usually account for unwanted properties including toxicity. Among strategies to profile small molecule-interacting proteins, photoaffinity labeling has been widely used because of its distinct advantages such as sensitivity. When combined with mass spectrometry, this approach can provide additional structural and mechanistic information, such as drug-target stoichiometry and exact interacting amino acid residues. We have described a novel fluorous photoaffinity labeling approach, in which a fluorous tag is incorporated into the photoaffinity labeling reagent to enable the enrichment of the labeled species from complex mixtures for analysis. This new feature likely makes the fluorous photoaffinity labeling approach suitable to identify transient interactions, and low-abundant, low-affinity interacting proteins in a cellular environment. PMID:25618351

  3. Labeling lake water with tritium

    USGS Publications Warehouse

    Frederick, B.J.

    1963-01-01

    A method of packaging tritiated water in a manner that facilitates safe handling in environmental labeling operations, and procedures followed in labeling a large body of water with a small volume of tritiated water are described. ?? 1963.

  4. 99m tc labeled liposomes

    SciTech Connect

    Phillips, W.T.; Klipper, R.W.; Timmons, J.H.; Rudolph, A.S.

    1992-10-27

    This patent describes a method of preparing stable gamma-emitting radionuclide-labeled alkyleneamine oxime, the incubating being for a period of time sufficient to form labeled liposome-encapsulated protein.

  5. Decode the Sodium Label Lingo

    MedlinePlus

    ... For Preschooler For Gradeschooler For Teen Decode the Sodium Label Lingo Published January 24, 2013 Print Email Reading food labels can help you slash sodium. Here's how to decipher them. "Sodium free" or " ...

  6. Use of indium-111 as a red cell label

    SciTech Connect

    AuBuchon, J.P.; Brightman, A.

    1989-02-01

    To select the most promising 111In chelate for use as a second red cell (RBC) label for comparison of the survival of autologous and allogeneic cells, 49 normal RBC samples were studied in vitro after being labeled with 111In-8-hydroxyquinolinol (111In-oxine) prepared by three different methods, 111In-tropolone, and 111In-acetylacetone. Labeling efficiencies reached 99 percent and did not decline when the amount of 111In used was increased from 1.75 to 50 muCi per ml of RBCs. Storage of labeled RBCs in normal AB plasma at 4, 22, and 37 degrees C for up to 48 hours resulted in a similar rate of loss of the label from the RBCs with all labeling methods. These rates were time- and temperature-dependent and were accurate predictions of the rates found in later in vivo experimentation. Fresh RBCs from 11 subjects were labeled with 111In chelated with oxine in the presence of the RBCs or chelated with tropolone just prior to the labeling. RBC mass determinations using these autologous RBCs labeled with 111In accurately reflected the subjects' RBC masses as predicted through standard morphometric formulae. The rate of disappearance of the radionuclide after reinfusion of the autologous RBCs decreased with time. At 24 hours after reinfusion, 89.5 +/- 1.29 percent (mean +/- SEM) of the 111In-tropolone and 87.3 +/- 1.25 percent of the 111In-oxine continued in circulation. 111In is a simple and efficient agent for the labeling of RBCs for blood volume determinations and short-term survivals.

  7. Benchmarking stable isotope labeling based quantitative proteomics.

    PubMed

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

    2013-08-01

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

  8. Learning with imperfectly labeled patterns

    NASA Technical Reports Server (NTRS)

    Chittineni, C. B.

    1979-01-01

    The problem of learning in pattern recognition using imperfectly labeled patterns is considered. The performance of the Bayes and nearest neighbor classifiers with imperfect labels is discussed using a probabilistic model for the mislabeling of the training patterns. Schemes for training the classifier using both parametric and non parametric techniques are presented. Methods for the correction of imperfect labels were developed. To gain an understanding of the learning process, expressions are derived for success probability as a function of training time for a one dimensional increment error correction classifier with imperfect labels. Feature selection with imperfectly labeled patterns is described.

  9. Glycan labeling strategies and their use in identification and quantification

    PubMed Central

    Ruhaak, L. R.; Zauner, G.; Huhn, C.; Bruggink, C.; Deelder, A. M.

    2010-01-01

    Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed. Figure MALDI-FTICR-MS of 2AA-labeled total plasma N-glycans PMID:20225063

  10. Intact Protein Quantitation Using Pseudoisobaric Dimethyl Labeling.

    PubMed

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

    2016-07-19

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

  11. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ....12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must...) If installation instructions are included on the label or with the package, add this statement:...

  12. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ....12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must...) If installation instructions are included on the label or with the package, add this statement:...

  13. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ....12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must...) If installation instructions are included on the label or with the package, add this statement:...

  14. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ....12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must...) If installation instructions are included on the label or with the package, add this statement:...

  15. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ....12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must...) If installation instructions are included on the label or with the package, add this statement:...

  16. Neutron Encoded Labeling for Peptide Identification

    PubMed Central

    Rose, Christopher M.; Merrill, Anna E.; Bailey, Derek J.; Hebert, Alexander S.; Westphall, Michael S.; Coon, Joshua J.

    2013-01-01

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, “Amino Acid Counter”, which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis. PMID:23638792

  17. Neutron encoded labeling for peptide identification.

    PubMed

    Rose, Christopher M; Merrill, Anna E; Bailey, Derek J; Hebert, Alexander S; Westphall, Michael S; Coon, Joshua J

    2013-05-21

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, "Amino Acid Counter", which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis. PMID:23638792

  18. Labeling and Identification of Direct Kinase Substrates

    PubMed Central

    Carlson, Scott M.; White, Forest M.

    2013-01-01

    Identifying kinase substrates is an important step in mapping signal transduction pathways, but remains a difficult and time-consuming process. Analog-sensitive kinases (AS-kinases) have been used to selectively tag and identify direct kinase substrates in lysates from whole cells. In this approach a gamma-thiol ATP-analog and AS-kinase are used to selectively thiophosphorylate target proteins. Thiophosphate is used as a chemical handle to purify peptides from a tryptic digest, and target proteins are identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we describe an updated strategy for labeling AS-kinase substrates, solid-phase capture of thiophosphorylated peptides, incorporation of stable-isotopic labeling in cell culture (SILAC) for filtering nonspecific background peptides, enrichment of phosphorylated target peptides to identify low-abundance targets, and analysis by LC-MS/MS. PMID:22669844

  19. 9 CFR 412.1 - Label approval.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    .... Jan. 6, 2014) § 412.1 Label approval. (a) No final label may be used on any product unless the label... for a corporation may submit only one label application for a product produced in other establishments...) The proposed label would not misrepresent the product; (ii) The use of the label would not present...

  20. Supplementing National Menu Labeling

    PubMed Central

    White, Lexi C.

    2012-01-01

    The US Food and Drug Administration’s forthcoming national menu labeling regulations are designed to help curb the national obesity epidemic by requiring calorie counts on restaurants’ menus. However, posted calories can be easily ignored or misunderstood by consumers and fail to accurately describe the healthiness of foods. We propose supplemental models that include nutritional information (e.g., fat, salt, sugar) or specific guidance (e.g., “heart-healthy” graphics). The goal is to empower restaurant patrons with better data to make healthier choices, and ultimately to reduce obesity prevalence. PMID:23078494

  1. Supplementing national menu labeling.

    PubMed

    Hodge, James G; White, Lexi C

    2012-12-01

    The US Food and Drug Administration's forthcoming national menu labeling regulations are designed to help curb the national obesity epidemic by requiring calorie counts on restaurants' menus. However, posted calories can be easily ignored or misunderstood by consumers and fail to accurately describe the healthiness of foods. We propose supplemental models that include nutritional information (e.g., fat, salt, sugar) or specific guidance (e.g., "heart-healthy" graphics). The goal is to empower restaurant patrons with better data to make healthier choices, and ultimately to reduce obesity prevalence. PMID:23078494

  2. Label and Label-Free Detection Techniques for Protein Microarrays

    PubMed Central

    Syahir, Amir; Usui, Kenji; Tomizaki, Kin-ya; Kajikawa, Kotaro; Mihara, Hisakazu

    2015-01-01

    Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano-biological events.

  3. Co-Labeling for Multi-View Weakly Labeled Learning.

    PubMed

    Xu, Xinxing; Li, Wen; Xu, Dong; Tsang, Ivor W

    2016-06-01

    It is often expensive and time consuming to collect labeled training samples in many real-world applications. To reduce human effort on annotating training samples, many machine learning techniques (e.g., semi-supervised learning (SSL), multi-instance learning (MIL), etc.) have been studied to exploit weakly labeled training samples. Meanwhile, when the training data is represented with multiple types of features, many multi-view learning methods have shown that classifiers trained on different views can help each other to better utilize the unlabeled training samples for the SSL task. In this paper, we study a new learning problem called multi-view weakly labeled learning, in which we aim to develop a unified approach to learn robust classifiers by effectively utilizing different types of weakly labeled multi-view data from a broad range of tasks including SSL, MIL and relative outlier detection (ROD). We propose an effective approach called co-labeling to solve the multi-view weakly labeled learning problem. Specifically, we model the learning problem on each view as a weakly labeled learning problem, which aims to learn an optimal classifier from a set of pseudo-label vectors generated by using the classifiers trained from other views. Unlike traditional co-training approaches using a single pseudo-label vector for training each classifier, our co-labeling approach explores different strategies to utilize the predictions from different views, biases and iterations for generating the pseudo-label vectors, making our approach more robust for real-world applications. Moreover, to further improve the weakly labeled learning on each view, we also exploit the inherent group structure in the pseudo-label vectors generated from different strategies, which leads to a new multi-layer multiple kernel learning problem. Promising results for text-based image retrieval on the NUS-WIDE dataset as well as news classification and text categorization on several real-world multi

  4. Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction.

    PubMed

    Gaudin, Zachary; Cerveau, Delphine; Marnet, Nathalie; Bouchereau, Alain; Delavault, Philippe; Simier, Philippe; Pouvreau, Jean-Bernard

    2014-01-21

    An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa. PMID:24359440

  5. Isotope Labeling Study of Retinal Chromophore Fragmentation.

    PubMed

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

    2016-04-28

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

  6. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... limited types of labels (e.g., labels for raw, single ingredient meat and poultry products) (48 FR 11410... poultry products will take effect January 1, 2012 (75 FR 82148, Dec. 29, 2010). These mandatory features... Agency. On March 25, 1992, FSIS published an Advance Notice of Proposed Rulemaking (ANPRM) (57 FR...

  7. Laser labeling, a safe technology to label produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Laser labeling of fruits and vegetables is an alternative means to label produce. Low energy CO2 laser beams etch the surface showing the contrasting underlying layer. These etched surfaces can promote water loss and potentially allow for entry of decay organisms. The long-term effects of laser labe...

  8. Laser labeling, a safe technology to label produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Labeling of the produce has gained marked attention in recent years. Laser labeling technology involves the etching of required information on the surface using a low energy CO2 laser beam. The etching forms alphanumerical characters by pinhole dot matrix depressions. These openings can lead to wat...

  9. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ... the Agency (76 FR 75809). FSIS also proposed to combine the regulations that provide for the approval... preamble (76 FR 75814), FSIS wrote: . . . statements on labels that are defined in FSIS's regulations or... ``Product Labeling: Definition of the Term ``Natural'' and related materials (71 FR 70503, Dec. 5, 2006)...

  10. Labeled Cocaine Analogs

    DOEpatents

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-03-30

    Novel methods for positron emission tomography or single photon emission spectroscopy using tracer compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)napthyl Y in .beta. configuration is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, The compounds bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

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

    PubMed

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

    2015-01-01

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

  12. Nutrition Marketing on Food Labels

    ERIC Educational Resources Information Center

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  13. Meat and Poultry Labeling Terms

    MedlinePlus

    ... Food Standards and Labels: The Facts Labeling and Marketing Information [ Top of Page ] OVEN PREPARED: Product is fully cooked and ready to eat. [ Top of Page ] YOUNG TURKEY: Turkeys of either sex that are less than 8 months of age according to present regulations. [ Top of Page ] Last ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Measurement of deuterium-labeled phylloquinone in plasma by LC-APCI-MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deuterium-labeled vegetables were fed to humans for the measurement of both unlabeled and deuterium-labeled phylloquinone in plasma. We developed a technique to determine the quantities of these compounds using liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC...

  16. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  17. 21 CFR 201.70 - Calcium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Calcium labeling. 201.70 Section 201.70 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.70 Calcium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the calcium content...

  18. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  19. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  20. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  1. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  2. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  3. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  4. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  5. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  6. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  7. Synthesis Of Labeled Metabolites

    DOEpatents

    Martinez, Rodolfo A.; Silks, III, Louis A.; Unkefer, Clifford J.; Atcher, Robert

    2004-03-23

    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  8. Labeled Cocaine Analogs

    DOEpatents

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-01-26

    Novel compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)naphthyl Y in .beta. configuration is Y.sub.1 or Y.sub.2, where Y.sub.1 is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, and Y.sub.2 is 2-methanesulfonyloxy ethoxy, 3-methanesulfonyloxy propoxy, 4-methanesulfonyloxy butoxy, 2-methanesulfonyloxy cyclopropoxy, 2 or 3-methanesulfonyloxy cyclobutoxy, 1'methanesulfonyloxy isopropoxy, 1'-fluoro, 3'-methanesulfonyloxy isopropoxy, 1'-methanesulfonyloxy, 3'-fluoro isopropoxy, 1'-methanesulfonyloxy isobutoxy, or 4'-methanesulfonyloxy isobutoxy bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  9. 27 CFR 19.517 - Statements required on labels under an exemption from label approval.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... labels under an exemption from label approval. 19.517 Section 19.517 Alcohol, Tobacco Products and... PLANTS Liquor Bottle, Label, and Closure Requirements Labeling Requirements § 19.517 Statements required on labels under an exemption from label approval. If a proprietor bottles spirits for domestic...

  10. 27 CFR 19.517 - Statements required on labels under an exemption from label approval.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... labels under an exemption from label approval. 19.517 Section 19.517 Alcohol, Tobacco Products and... PLANTS Liquor Bottle, Label, and Closure Requirements Labeling Requirements § 19.517 Statements required on labels under an exemption from label approval. If a proprietor bottles spirits for domestic...

  11. 27 CFR 19.517 - Statements required on labels under an exemption from label approval.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... labels under an exemption from label approval. 19.517 Section 19.517 Alcohol, Tobacco Products and... PLANTS Liquor Bottle, Label, and Closure Requirements Labeling Requirements § 19.517 Statements required on labels under an exemption from label approval. If a proprietor bottles spirits for domestic...

  12. 27 CFR 19.517 - Statements required on labels under an exemption from label approval.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... labels under an exemption from label approval. 19.517 Section 19.517 Alcohol, Tobacco Products and... PLANTS Liquor Bottle, Label, and Closure Requirements Labeling Requirements § 19.517 Statements required on labels under an exemption from label approval. If a proprietor bottles spirits for domestic...

  13. Photoactivatable protein labeling by singlet oxygen mediated reactions.

    PubMed

    To, Tsz-Leung; Medzihradszky, Katalin F; Burlingame, Alma L; DeGrado, William F; Jo, Hyunil; Shu, Xiaokun

    2016-07-15

    Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling. PMID:27220724

  14. SU-E-I-14: Comparison of Iodine-Labeled and Indium-Labeled Antibody Biodistributions

    SciTech Connect

    Williams, L

    2014-06-01

    Purpose: It is often assumed that animal biodistributions of novel proteins are not dependent upon the radiolabel used in their determination. In units of percent injected dose per gram of tissue (%ID/g), organ uptake results (u) may be obtained using either iodine or metal as radioactive labels. Iodination is preferred as it is a one-step process whereas metal labeling requires two chemical procedures and therefore more protein material. It is important to test whether the radioactive tag leads to variation in the uptake value. Methods: Uptakes of 3antibodies to Carcinoembryonic Antigen (CEA) were evaluated in a nude mouse model bearing 150 to 300 mg LS174T human colon cancer xenografts. Antibodies included diabody (56 kDa), minibody (80kDa) and intact M5A (150 kDa) anti-CEA cognates. Both radioiodine and indium-111 labels were used with uptakes evaluated at 7 time(t) points out to 96 h. Ratios (R) of u(iodine-label)/u(indium-label) were determined for liver, spleen, kidneys, lung and tumor. Results: Hepatic loss was rapid for diabody and minibody; by 24 h their R values were only 2%; i.e., uptake of iodine was 2% of that of indium for these 2 antibodies. By contrast, R for the intact cognate was 50% at that time point. Splenic results were similar. Tumor uptake ratios did not depend upon the antibody type and were 50% at 24 h. Conclusions: Relatively rapid loss of iodine relative to indium in liver and spleen was observed in lower mass antibodies. Tumor ratios were larger and independent of antibody type. Aside from tumor, the R ratio of uptakes depended on the antibody type. R values decreased monotonically with time in all tissues and for all cognates. Using this ratio, one can possibly correct iodine-based u (t) results so that they resemble radiometal-derived biodistributions.

  15. Selective chemical labeling of proteins.

    PubMed

    Chen, Xi; Wu, Yao-Wen

    2016-06-28

    Over the years, there have been remarkable efforts in the development of selective protein labeling strategies. In this review, we deliver a comprehensive overview of the currently available bioorthogonal and chemoselective reactions. The ability to introduce bioorthogonal handles to proteins is essential to carry out bioorthogonal reactions for protein labeling in living systems. We therefore summarize the techniques that allow for site-specific "installation" of bioorthogonal handles into proteins. We also highlight the biological applications that have been achieved by selective chemical labeling of proteins. PMID:26940577

  16. Mass Spec Studio for Integrative Structural Biology

    PubMed Central

    Rey, Martial; Sarpe, Vladimir; Burns, Kyle; Buse, Joshua; Baker, Charles A.H.; van Dijk, Marc; Wordeman, Linda; Bonvin, Alexandre M.J.J.; Schriemer, David C.

    2015-01-01

    SUMMARY The integration of biophysical data from multiple sources is critical for developing accurate structural models of large multiprotein systems and their regulators. Mass spectrometry (MS) can be used to measure the insertion location for a wide range of topographically sensitive chemical probes, and such insertion data provide a rich, but disparate set of modeling restraints. We have developed a software platform that integrates the analysis of label-based MS data with protein modeling activities (Mass Spec Studio). Analysis packages can mine any labeling data from any mass spectrometer in a proteomics-grade manner, and link labeling methods with data-directed protein interaction modeling using HADDOCK. Support is provided for hydrogen/ deuterium exchange (HX) and covalent labeling chemistries, including novel acquisition strategies such as targeted HX-tandem MS (MS2) and data-independent HX-MS2. The latter permits the modeling of highly complex systems, which we demonstrate by the analysis of microtubule interactions. PMID:25242457

  17. How to read food labels

    MedlinePlus

    ... 1 serving. You should also pay attention to trans fats on any food label. These fats raise "bad" ... foods and desserts. Many fast food restaurants use trans fats for frying. If a food has these fats, ...

  18. Dietary Supplement Label Database (DSLD)

    MedlinePlus

    ... Print Report Error T he Dietary Supplement Label Database (DSLD) is a joint project of the National ... participants in the latest survey in the DSLD database (NHANES): The search options: Quick Search, Browse Dietary ...

  19. Food Labels Tell the Story!

    MedlinePlus

    ... Environment Kids Health Topics Environment & Health Healthy Living Pollution Reduce, Reuse, Recycle Science – How It Works The ... Pay close attention to serving sizes. Products labeled "light" or "lite" must have 1/3 fewer calories ...

  20. Differential labeling of free and disulfide-bound thiol functions in proteins.

    PubMed

    Seiwert, Bettina; Hayen, Heiko; Karst, Uwe

    2008-01-01

    A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid chromatography/electrochemistry/mass spectrometry was used to assign the N-(2-ferroceneethyl)maleimide (FEM) labeled free cysteine functionalities in a tryptic digest mixture, whereas a precursor ion scan enables the detection of peptides with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) labeled disulfide-bound cysteine groups after reduction. Fragment spectra of the labeled peptides yield an excellent coverage of b-type and y-type ions. The ferrocene labeled cysteines were fragmented as 412 Da (FEM) and 455 Da (FMEA). These fragment masses are significantly higher than unlabeled amino acids or dipeptides and are easily detected. The position of free and disulfide-bound cysteine may therefore be assigned in an amino acid sequence. PMID:17977013

  1. The impact of nutritional labels and socioeconomic status on energy intake. An experimental field study.

    PubMed

    Crockett, Rachel A; Jebb, Susan A; Hankins, Matthew; Marteau, Theresa M

    2014-10-01

    There is some evidence for paradoxical effects of nutritional labelling on energy intake particularly amongst restrained eaters and those with a higher body mass index (BMI) resulting in greater consumption of energy from foods with a positive health message (e.g. "low-fat") compared with the same foods, unlabelled. This study aimed to investigate, in a UK general population sample, the likelihood of paradoxical effects of nutritional labelling on energy intake. Participants (n = 287) attended a London cinema and were offered a large tub of salted or toffee popcorn. Participants were randomised to receive their selected flavour with one of three labels: a green low-fat label, a red high-fat label or no label. Participants watched two film clips while completing measures of demographic characteristics, emotional state and taste of the popcorn. Following the experiment, popcorn consumption was measured. There were no main effects of nutritional labelling on consumption. Contrary to predictions neither BMI nor weight concern moderated the effect of label on consumption. There was a three-way interaction between low-fat label, weight concern and socioeconomic status (SES) such that weight-concerned participants of higher SES who saw a low-fat label consumed more than weight unconcerned participants of similar SES (t = -2.7, P = .04). By contrast, weight-concerned participants of lower SES seeing either type of label, consumed less than those seeing no label (t = -2.04, P = .04). Nutritional labelling may have different effects in different socioeconomic groups. Further studies are required to understand fully the possible contribution of food labelling to health inequalities. PMID:24879885

  2. Parallel Reaction Monitoring: A Targeted Experiment Performed Using High Resolution and High Mass Accuracy Mass Spectrometry

    PubMed Central

    Rauniyar, Navin

    2015-01-01

    The parallel reaction monitoring (PRM) assay has emerged as an alternative method of targeted quantification. The PRM assay is performed in a high resolution and high mass accuracy mode on a mass spectrometer. This review presents the features that make PRM a highly specific and selective method for targeted quantification using quadrupole-Orbitrap hybrid instruments. In addition, this review discusses the label-based and label-free methods of quantification that can be performed with the targeted approach. PMID:26633379

  3. LabeledIn: cataloging labeled indications for human drugs.

    PubMed

    Khare, Ritu; Li, Jiao; Lu, Zhiyong

    2014-12-01

    Drug-disease treatment relationships, i.e., which drug(s) are indicated to treat which disease(s), are among the most frequently sought information in PubMed®. Such information is useful for feeding the Google Knowledge Graph, designing computational methods to predict novel drug indications, and validating clinical information in EMRs. Given the importance and utility of this information, there have been several efforts to create repositories of drugs and their indications. However, existing resources are incomplete. Furthermore, they neither label indications in a structured way nor differentiate them by drug-specific properties such as dosage form, and thus do not support computer processing or semantic interoperability. More recently, several studies have proposed automatic methods to extract structured indications from drug descriptions; however, their performance is limited by natural language challenges in disease named entity recognition and indication selection. In response, we report LabeledIn: a human-reviewed, machine-readable and source-linked catalog of labeled indications for human drugs. More specifically, we describe our semi-automatic approach to derive LabeledIn from drug descriptions through human annotations with aids from automatic methods. As the data source, we use the drug labels (or package inserts) submitted to the FDA by drug manufacturers and made available in DailyMed. Our machine-assisted human annotation workflow comprises: (i) a grouping method to remove redundancy and identify representative drug labels to be used for human annotation, (ii) an automatic method to recognize and normalize mentions of diseases in drug labels as candidate indications, and (iii) a two-round annotation workflow for human experts to judge the pre-computed candidates and deliver the final gold standard. In this study, we focused on 250 highly accessed drugs in PubMed Health, a newly developed public web resource for consumers and clinicians on prevention

  4. Multi-focus cluster labeling.

    PubMed

    Eikvil, Line; Jenssen, Tor-Kristian; Holden, Marit

    2015-06-01

    Document collections resulting from searches in the biomedical literature, for instance, in PubMed, are often so large that some organization of the returned information is necessary. Clustering is an efficient tool for organizing search results. To help the user to decide how to continue the search for relevant documents, the content of each cluster can be characterized by a set of representative keywords or cluster labels. As different users may have different interests, it can be desirable with solutions that make it possible to produce labels from a selection of different topical categories. We therefore introduce the concept of multi-focus cluster labeling to give users the possibility to get an overview of the contents through labels from multiple viewpoints. The concept for multi-focus cluster labeling has been established and has been demonstrated on three different document collections. We illustrate that multi-focus visualizations can give an overview of clusters along axes that general labels are not able to convey. The approach is generic and should be applicable to any biomedical (or other) domain with any selection of foci where appropriate focus vocabularies can be established. A user evaluation also indicates that such a multi-focus concept is useful. PMID:25869415

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

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  6. Isobaric Labeling-Based Relative Quantification in Shotgun Proteomics

    PubMed Central

    2015-01-01

    Mass spectrometry plays a key role in relative quantitative comparisons of proteins in order to understand their functional role in biological systems upon perturbation. In this review, we review studies that examine different aspects of isobaric labeling-based relative quantification for shotgun proteomic analysis. In particular, we focus on different types of isobaric reagents and their reaction chemistry (e.g., amine-, carbonyl-, and sulfhydryl-reactive). Various factors, such as ratio compression, reporter ion dynamic range, and others, cause an underestimation of changes in relative abundance of proteins across samples, undermining the ability of the isobaric labeling approach to be truly quantitative. These factors that affect quantification and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements will be discussed. Finally, the extended application of isobaric labeling-based approach in hyperplexing strategy, targeted quantification, and phosphopeptide analysis are also examined. PMID:25337643

  7. 49 CFR 172.426 - OXIDIZER label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... SECURITY PLANS Labeling § 172.426 OXIDIZER label. (a) Except for size and color, the OXIDIZER label must be as follows: EC02MR91.027 (b) In addition to complying with § 172.407, the background color on the OXIDIZER label must be yellow....

  8. 49 CFR 172.426 - OXIDIZER label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... SECURITY PLANS Labeling § 172.426 OXIDIZER label. (a) Except for size and color, the OXIDIZER label must be as follows: EC02MR91.027 (b) In addition to complying with § 172.407, the background color on the OXIDIZER label must be yellow....

  9. 49 CFR 172.426 - OXIDIZER label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SECURITY PLANS Labeling § 172.426 OXIDIZER label. (a) Except for size and color, the OXIDIZER label must be as follows: EC02MR91.027 (b) In addition to complying with § 172.407, the background color on the OXIDIZER label must be yellow....

  10. 49 CFR 172.426 - OXIDIZER label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... SECURITY PLANS Labeling § 172.426 OXIDIZER label. (a) Except for size and color, the OXIDIZER label must be as follows: EC02MR91.027 (b) In addition to complying with § 172.407, the background color on the OXIDIZER label must be yellow....

  11. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall appear on the label affixed to each package containing a product: (a) The proper name of the product; (b... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Package label. 610.61 Section 610.61 Food...

  12. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  13. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  14. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  15. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall appear on the label affixed to each package containing a product: (a) The proper name of the product; (b... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Package label. 610.61 Section 610.61 Food...

  16. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall appear on the label affixed to each package containing a product: (a) The proper name of the product; (b) The... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Package label. 610.61 Section 610.61 Food and...

  17. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  18. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall appear on the label affixed to each package containing a product: (a) The proper name of the product; (b... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Package label. 610.61 Section 610.61 Food...

  19. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall appear on the label affixed to each package containing a product: (a) The proper name of the product; (b... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Package label. 610.61 Section 610.61 Food...

  20. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  1. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... THAT IT IS REMANUFACTURED, EXCEPT AS ALLOWED BY 40 CFR 1033.750.” (3) Label diesel-fueled locomotives... that contrasts with the background of the label. (iii) The label must include all the following... same engine part. (ii) The label must be lettered in the English language using a color that...

  2. 40 CFR 156.10 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Labeling requirements. 156.10 Section 156.10 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS LABELING REQUIREMENTS FOR PESTICIDES AND DEVICES General Provisions § 156.10 Labeling requirements. (a) General—(1) Contents of the label. Every...

  3. 30 CFR 74.15 - Approval labels.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Approval labels. 74.15 Section 74.15 Mineral... DUST SAMPLING DEVICES General Requirements for All Devices § 74.15 Approval labels. (a) Certificate of... reproductions of approval labels and a sketch or description of the position of the labels on each...

  4. 16 CFR 306.12 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 1 2012-01-01 2012-01-01 false Labels. 306.12 Section 306.12 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS AUTOMOTIVE FUEL RATINGS, CERTIFICATION AND POSTING Label Specifications § 306.12 Labels. All labels must meet the following specifications: (a) Layout—(1) For gasoline...

  5. 16 CFR 306.12 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 1 2014-01-01 2014-01-01 false Labels. 306.12 Section 306.12 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS AUTOMOTIVE FUEL RATINGS, CERTIFICATION AND POSTING Label Specifications § 306.12 Labels. All labels must meet the following specifications: (a) Layout—(1) For gasoline...

  6. Quantification of active infliximab in human serum with liquid chromatography-tandem mass spectrometry using a tumor necrosis factor alpha -based pre-analytical sample purification and a stable isotopic labeled infliximab bio-similar as internal standard: A target-based, sensitive and cost-effective method.

    PubMed

    El Amrani, Mohsin; van den Broek, Marcel P H; Göbel, Camiel; van Maarseveen, Erik M

    2016-07-01

    The therapeutic monoclonal antibody Infliximab (IFX) is a widely used drug for the treatment of several inflammatory autoimmune diseases. However, approximately 10% of patients develop anti-infliximab antibodies (ATIs) rendering the treatment ineffective. Early detection of underexposure to unbound IFX would result in a timely switch of therapy which could aid in the treatment of this disease. Streptavidin coated 96 well plates were used to capture biotinylated-tumor necrosis factor -alpha (b-TNF-α), which in turn was used to selectively extract the active form of IFX in human serum. After elution, IFX was digested using trypsin and one signature peptide was selected for subsequent analysis on liquid chromatography-tandem mass spectrometry (LC-MS/MS). The internal standard used was a stable isotopic labeled IFX bio-similar. The assay was successfully validated according to European Medicines Agency (EMA) guidelines and was found to be linear in a range of 0.5-20μg/mL (r(2)=0.994). Lower limit of quantification for the assay (<20% CV) was 0.5μg/mL, requiring only 2μL of sample. Cross-validation against enzyme-linked immunosorbent assay (ELISA) resulted in a high correlation between methods (r(2)=0.95 with a ρc=0.83) and the accuracy was in line with previously published results. In conclusion, a sensitive, robust and cost-effective method was developed for the bio-analysis of IFX with LC-MS/MS by means of a target-based pre-analytical sample purification. Moreover, low volume and costs of consumables per sample promote its feasibility in (pre)clinical studies and in therapeutic drug monitoring. This method should be considered as first choice due to its accuracy and multiple degree of selectivity. PMID:27264745

  7. Nutrition Labeling Using a Computer Program

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    The 1990 Nutrition Labeling and Education Act mandated nutritional labeling of most foods. As a result, a large portion of food analysis is performed for nutritional labeling purposes. A food labeling guide and links to the complete nutritional labeling regulations are available online at http://vm.cfsan.fda.gov/˜dms/flg-toc.html. However, interpretation of these regulations and the appropriate usage of rounding rules, available nutrient content claims, reference amounts, and serving size can be difficult.

  8. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 2 2011-10-01 2011-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  9. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  10. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 2 2013-10-01 2013-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  11. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 2 2014-10-01 2014-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  12. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 2 2012-10-01 2012-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  13. Labeling Feral Spruce Budworm (Lepidoptera: Tortricidae) Populations With Rubidium.

    PubMed

    MacKinnon, Wayne; Eveleigh, Eldon; Silk, Peter; Forbes, Glen

    2016-04-01

    Rubidium (Rb) is a trace element that occurs naturally in low concentrations and is easily absorbed by plants, making it a useful tool for labeling insect defoliators, such as spruce budworm (Choristoneura fumiferana Clemens). Balsam fir trees (Abies balsamea (L.) Miller) injected with either 8 or 16 g per tree of rubidium chloride (RbCl) showed quick uptake and distribution throughout the crown, with no negative effects on tree shoot growth or spruce budworm survival and development. Adult spruce budworm that fed as larvae on trees injected with RbCl were clearly labeled, with significantly higher Rb concentrations than the background levels found in adults that fed as larvae on control trees. Rb concentrations in feral spruce budworm adults for both the 8 g (9 µg/g) and 16 g (25 µg/g) per tree treatments were at least five times lower than those in laboratory-reared adults on 1,000 µg/g RbCl diet (125 µg/g); survival, development, pupal weight, sex ratio, and mating status of spruce budworm were not adversely affected by Rb treatment. Egg masses laid by feral females that fed as larvae on Rb-labeled trees were also labeled with Rb. Injecting trees with RbCl is a viable technique for labeling feral spruce budworm populations to help distinguish local populations from immigrants to better evaluate the success of early intervention strategies such as mating disruption. PMID:26920559

  14. Galaxy masses

    NASA Astrophysics Data System (ADS)

    Courteau, Stéphane; Cappellari, Michele; de Jong, Roelof S.; Dutton, Aaron A.; Emsellem, Eric; Hoekstra, Henk; Koopmans, L. V. E.; Mamon, Gary A.; Maraston, Claudia; Treu, Tommaso; Widrow, Lawrence M.

    2014-01-01

    Galaxy masses play a fundamental role in our understanding of structure formation models. This review addresses the variety and reliability of mass estimators that pertain to stars, gas, and dark matter. The different sections on masses from stellar populations, dynamical masses of gas-rich and gas-poor galaxies, with some attention paid to our Milky Way, and masses from weak and strong lensing methods all provide review material on galaxy masses in a self-consistent manner.

  15. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  16. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  17. Label-free drug discovery

    PubMed Central

    Fang, Ye

    2014-01-01

    Current drug discovery is dominated by label-dependent molecular approaches, which screen drugs in the context of a predefined and target-based hypothesis in vitro. Given that target-based discovery has not transformed the industry, phenotypic screen that identifies drugs based on a specific phenotype of cells, tissues, or animals has gained renewed interest. However, owing to the intrinsic complexity in drug–target interactions, there is often a significant gap between the phenotype screened and the ultimate molecular mechanism of action sought. This paper presents a label-free strategy for early drug discovery. This strategy combines label-free cell phenotypic profiling with computational approaches, and holds promise to bridge the gap by offering a kinetic and holistic representation of the functional consequences of drugs in disease relevant cells that is amenable to mechanistic deconvolution. PMID:24723889

  18. Metrics for Labeled Markov Systems

    NASA Technical Reports Server (NTRS)

    Desharnais, Josee; Jagadeesan, Radha; Gupta, Vineet; Panangaden, Prakash

    1999-01-01

    Partial Labeled Markov Chains are simultaneously generalizations of process algebra and of traditional Markov chains. They provide a foundation for interacting discrete probabilistic systems, the interaction being synchronization on labels as in process algebra. Existing notions of process equivalence are too sensitive to the exact probabilities of various transitions. This paper addresses contextual reasoning principles for reasoning about more robust notions of "approximate" equivalence between concurrent interacting probabilistic systems. The present results indicate that:We develop a family of metrics between partial labeled Markov chains to formalize the notion of distance between processes. We show that processes at distance zero are bisimilar. We describe a decision procedure to compute the distance between two processes. We show that reasoning about approximate equivalence can be done compositionally by showing that process combinators do not increase distance. We introduce an asymptotic metric to capture asymptotic properties of Markov chains; and show that parallel composition does not increase asymptotic distance.

  19. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  20. Negative mass

    NASA Astrophysics Data System (ADS)

    Hammond, Richard T.

    2015-03-01

    Some physical aspects of negative mass are examined. Several unusual properties, such as the ability of negative mass to penetrate any armor, are analysed. Other surprising effects include the bizarre system of negative mass chasing positive mass, naked singularities and the violation of cosmic censorship, wormholes, and quantum mechanical results as well. In addition, a brief look into the implications for strings is given.

  1. The Labelling Approach to Deviance.

    ERIC Educational Resources Information Center

    Rains, Prudence M.; Kitsuse, John L.; Duster, Troy; Freidson, Eliot

    2003-01-01

    This reprint of one chapter from the 1975 text, "Issues in the Classification of Children" by Nicholas Hobbs and others, addresses the theoretical, methodological, and empirical issues involved in the "labeling" approach to the sociology of deviance. It examines the social process of classification, the use of classification in social agencies,…

  2. Revisiting Labels: "Hearing" or Not?

    ERIC Educational Resources Information Center

    Rhoades, Ellen A.

    2010-01-01

    This position paper briefly presents evidence-based findings pertaining to the language of labels for people with hearing loss that relate to stigma, expectation levels, stereotypes, and self-fulfilling prophecies. These constructs are important for auditory-based practitioners, administrators, policymakers, students, families, and persons with…

  3. Labeling: A Dilemma or Solution?

    ERIC Educational Resources Information Center

    Perusin, Adriano

    1998-01-01

    Reviews the pros and cons of labeling within the classroom, including the effects on self-concept and peer relationships. Argues that integration can be successful, if implemented by an effective and prepared instructor and that activities which are accommodating may allow integration to be successful. (Author/CR)

  4. When Diagnostic Labels Mask Trauma

    ERIC Educational Resources Information Center

    Foltz, Robert; Dang, Sidney; Daniels, Brian; Doyle, Hillary; McFee, Scott; Quisenberry, Carolyn

    2013-01-01

    A growing body of research shows that many seriously troubled children and adolescents are reacting to adverse life experiences. Yet traditional diagnostic labels are based on checklists of surface symptoms. Distracted by disruptive behavior, the common response is to medicate, punish, or exclude rather than respond to needs of youth who have…

  5. Nutrition Marketing on Food Labels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nutrition marketing may influence purchasing behavior and thereby be a factor in the obesity epidemic. Very little peer-reviewed research has been published which investigates the relationship between nutrition marketing on food labels and consumer behavior. The purpose of this paper was to give an ...

  6. Psychological effectiveness of carbon labelling

    NASA Astrophysics Data System (ADS)

    Beattie, Geoffrey

    2012-04-01

    Despite the decision by supermarket-giant Tesco to delay its plan to add carbon-footprint information onto all of its 70,000 products, carbon labelling, if carefully designed, could yet change consumer behaviour. However, it requires a new type of thinking about consumers and much additional work.

  7. The labeling debate in the United States.

    PubMed

    Marchant, Gary E; Cardineau, Guy A

    2013-01-01

    The mandatory labeling of genetically modified (GM) food has become the predominant policy issue concerning biotechnology in the United States. The controversy over GM labeling is being debated at several different levels and branches of government. At the federal level, the Food and Drug Administration, which has primary jurisdiction over food safety and labeling, has steadfastly refused to require labeling of GM foods since 1992 based on its conclusion that GM foods as a category present no unique or higher risks than other foods. Proposed legislation has been repeatedly introduced in the US. Congress over the years to mandate GM labeling, but has made very little progress. With federal labeling requirements apparently stalled, the main activity has switched to the state level, where numerous individual states are considering mandatory GM labeling, either through legislation or proposition. The debate over GM labeling, at both the federal and state levels, has focused on five issues: (1) public opinion; (2) the legality of labeling requirements; (3) the risks and benefits of GM foods; (4) the costs and burdens of GM labeling; and (5) consumer choice. While the pro-labeling forces argue that all of these factors weigh in favor of mandatory GM labeling, a more careful evaluation of the evidence finds that all five factors weigh decisively against mandatory GM labeling requirements. PMID:23982076

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

    PubMed Central

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

    2013-01-01

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

  9. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  10. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  11. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  12. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  13. 7 CFR 65.400 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... origin declarations can either be in the form of a placard, sign, label, sticker, band, twist tie, pin... declaration of the country of origin (e.g., placard, sign, label, sticker, band, twist tie, pin tag, or...

  14. 27 CFR 19.437 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... affix a label showing the following information: (1) The proprietor's name and plant number; (2) The... paragraph (a) of this section is not required when the sample container bears a label approved under part...

  15. 27 CFR 19.704 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... containers bear an approved label pursuant to 27 CFR Part 5 and subpart S of this part and the sample is... spirits to be withdrawn under the provisions of § 19.701, the proprietor shall affix a label showing...

  16. 27 CFR 19.437 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... affix a label showing the following information: (1) The proprietor's name and plant number; (2) The... paragraph (a) of this section is not required when the sample container bears a label approved under part...

  17. 27 CFR 19.437 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... affix a label showing the following information: (1) The proprietor's name and plant number; (2) The... paragraph (a) of this section is not required when the sample container bears a label approved under part...

  18. 27 CFR 19.437 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... affix a label showing the following information: (1) The proprietor's name and plant number; (2) The... paragraph (a) of this section is not required when the sample container bears a label approved under part...

  19. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  20. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... THAT IT IS REMANUFACTURED, EXCEPT AS ALLOWED BY 40 CFR 1033.750.” (3) Label diesel-fueled locomotives... locomotives certified for use with both LSD and ULSD. (c) Engine labels. (1) For engines not...

  1. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... THAT IT IS REMANUFACTURED, EXCEPT AS ALLOWED BY 40 CFR 1033.750.” (3) Label diesel-fueled locomotives... locomotives certified for use with both LSD and ULSD. (c) Engine labels. (1) For engines not...

  2. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... THAT IT IS REMANUFACTURED, EXCEPT AS ALLOWED BY 40 CFR 1033.750.” (3) Label diesel-fueled locomotives... locomotives certified for use with both LSD and ULSD. (c) Engine labels. (1) For engines not...

  3. Ivabradine: A Review of Labeled and Off-Label Uses.

    PubMed

    Oliphant, Carrie S; Owens, Ryan E; Bolorunduro, Oluwaseyi B; Jha, Sunil K

    2016-10-01

    Ivabradine is a unique medication recently approved in the USA for the treatment of select heart failure patients. It was first approved for use in several countries around the world over a decade ago as an anti-anginal agent, with subsequent approval for use in heart failure patients. Since ivabradine has selective activity blocking the I f currents in the sinus node, it can reduce heart rate without appreciable effects on blood pressure. Given this heart-rate-specific effect, it has been investigated in many off-label indications as an alternative to traditional heart-rate-reducing medications such as beta blockers and calcium channel blockers. We conducted searches of PubMed and Google Scholar for ivabradine, heart failure, HFrEF, HFpEF, angina, coronary artery disease, inappropriate sinus tachycardia, postural orthostatic hypotension, coronary computed tomography angiography and atrial fibrillation. We reviewed and included studies, case reports, and case series published between 1980 and June 2016 if they provided information relevant to the practicing clinician. In many cases, larger clinical trials are needed to solidify the benefit of ivabradine, although studies indicate benefit in most therapeutic areas explored to date. The purpose of this paper is to review the current labeled and off-label uses of ivabradine, with a focus on clinical trial data. PMID:27405864

  4. Mass loss

    NASA Technical Reports Server (NTRS)

    Goldberg, Leo

    1987-01-01

    Observational evidence for mass loss from cool stars is reviewed. Spectra line profiles are used for the derivation of mass-loss rates with the aid of the equation of continuity. This equation implies steady mass loss with spherical symmetry. Data from binary stars, Mira variables, and red giants in globular clusters are examined. Silicate emission is discussed as a useful indicator of mass loss in the middle infrared spectra. The use of thermal millimeter-wave radiation, Very Large Array (VLA) measurement of radio emission, and OH/IR masers are discussed as a tool for mass loss measurement. Evidence for nonsteady mass loss is also reviewed.

  5. Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling.

    PubMed

    Palmblad, Magnus; Mills, Davinia J; Bindschedler, Laurence V

    2008-02-01

    We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing. PMID:18189342

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

    PubMed Central

    2015-01-01

    Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X13CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X13CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X13CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X13CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X13CMS with an analysis of cultured rat astrocytes treated with uniformly

  7. Use of In-labeled autologous leukocytes to image an abdominal abscess in a horse

    SciTech Connect

    Koblik, P.D.; Lofstedt, J.; Jakowski, R.M.; Johnson, K.L.

    1985-06-15

    Indium 111-labeled autologous leukocytes were used to image an abdominal abscess in a horse with a palpable abdominal mass and history of Streptococcus equi infection. A focal area of radioactivity was identified in the location corresponding to the abscess. Imaging of this focal uptake was optimal 48 hours after injection. Similar scans obtained in 2 clinically normal horses revealed no evidence of focal radioactivity in this region. The cell labeling procedure gave acceptable labeling efficiency (87.5%) but an excessive number of damaged WBC, resulting in persistent lung radioactivity on all images. No adverse effects were noted. Radiation measured in the horse and its excreta were well within acceptable limits.

  8. 99mTc: Labeling Chemistry and Labeled Compounds

    NASA Astrophysics Data System (ADS)

    Alberto, R.; Abram, U.

    This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section

  9. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be...

  10. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be...

  11. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be...

  12. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  13. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  14. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  15. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  16. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  17. Learning Words from Labeling and Directive Speech

    ERIC Educational Resources Information Center

    Callanan, Maureen A.; Akhtar, Nameera; Sussman, Lisa

    2014-01-01

    Despite the common intuition that labeling may be the best way to teach a new word to a child, systematic testing is needed of the prediction that children learn words better from labeling utterances than from directive utterances. Two experiments compared toddlers' label learning in the context of hearing words used in directive versus labeling…

  18. China`s environmental labeling program

    SciTech Connect

    Zhao, J.; Xia, Q.

    1999-09-01

    China`s environmental labeling program was created because the government wanted to improve environmental management, and some Chinese enterprises expected that labeling would help eliminate non-tariff barriers for their exports and allow them to expand their domestic market shares. The labeling program, which is managed by the Chinese government, is a voluntary third-party certification system based on labeling procedures developed in Organization for Economic Co-operation and Development countries. Thus far, China`s labeling program has increased consumer awareness of labeled products and encouraged some enterprises to adopt cleaner technologies in products that have close relationships with consumers` health. However, the effectiveness of the program is very limited because only a small number of product categories are included in the program, consumers` purchasing behavior in China has been influenced by environmental labels for only several types of products, and relatively few enterprises participate in this program. The following measures would improve the effectiveness of the labeling program: enhancing public awareness of environmental labeling and environmental protection, increasing the number of product categories involved in environmental labeling, displaying more information on labels, and integrating the labeling program into China`s efforts to promote cleaner production and to encourage compliance with ISO 14000 standards.

  19. 21 CFR 660.55 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Labeling. 660.55 Section 660.55 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Anti-Human Globulin § 660.55 Labeling. In addition to the applicable labeling requirements...

  20. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... TREASURY ALCOHOL PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container of concentrate will have affixed thereto, before transfer, a label identifying the product and... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Label. 18.55 Section...

  1. 47 CFR 15.19 - Labelling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...), and the resulting product is not separately tested: ER09DE03.001 (2) Label text and information should... label on these products shall be permanently affixed to the product and shall be readily visible to the... label shall be located in a conspicuous location on the device and shall contain the...

  2. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... following items shall appear on the label affixed to each container of a product capable of bearing a full label: (1) The proper name of the product; (2) The name, address, and license number of manufacturer; (3... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Container label. 610.60 Section 610.60 Food...

  3. 47 CFR 15.19 - Labelling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...), and the resulting product is not separately tested: ER09DE03.001 (2) Label text and information should... label on these products shall be permanently affixed to the product and shall be readily visible to the... label shall be located in a conspicuous location on the device and shall contain the...

  4. 47 CFR 15.19 - Labelling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...), and the resulting product is not separately tested: ER09DE03.001 (2) Label text and information should... label on these products shall be permanently affixed to the product and shall be readily visible to the... label shall be located in a conspicuous location on the device and shall contain the...

  5. 27 CFR 19.604 - Caution label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Caution label. 19.604... OF THE TREASURY LIQUORS DISTILLED SPIRITS PLANTS Containers and Marks Marks § 19.604 Caution label... denaturer may be printed on such label, but no other extraneous matter will be permitted thereon without...

  6. 47 CFR 15.19 - Labelling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...), and the resulting product is not separately tested: ER09DE03.001 (2) Label text and information should... label on these products shall be permanently affixed to the product and shall be readily visible to the... label shall be located in a conspicuous location on the device and shall contain the...

  7. 40 CFR 763.171 - Labeling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... cannot be removed without defacing or destroying them. Product labels shall appear as in paragraph (d)(2... packaging, the label must be attached to the innermost layer adjacent to the product. If the innermost layer... product's innermost layer of product wrapping or packaging, or a label must be attached to the next...

  8. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  9. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... TREASURY ALCOHOL PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container of concentrate will have affixed thereto, before transfer, a label identifying the product and... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Label. 18.55 Section...

  10. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... following items shall appear on the label affixed to each container of a product capable of bearing a full label: (1) The proper name of the product; (2) The name, address, and license number of manufacturer; (3... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Container label. 610.60 Section 610.60 Food...

  11. 21 CFR 606.121 - Container label.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Container label. 606.121 Section 606.121 Food and... CURRENT GOOD MANUFACTURING PRACTICE FOR BLOOD AND BLOOD COMPONENTS Finished Product Control § 606.121 Container label. (a) The container label requirements are designed to facilitate the use of a...

  12. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container of concentrate will have affixed thereto, before transfer, a label identifying the product and... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Label. 18.55 Section...

  13. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... following items shall appear on the label affixed to each container of a product capable of bearing a full label: (1) The proper name of the product; (2) The name, address, and license number of manufacturer; (3... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Container label. 610.60 Section 610.60 Food...

  14. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    .... Each label shall be identified as to: (1) Name and product code number as it appears on the product... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Label records. 116.3 Section 116.3..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label...

  15. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  16. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... following items shall appear on the label affixed to each container of a product capable of bearing a full label: (1) The proper name of the product; (2) The name, address, and license number of manufacturer; (3... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Container label. 610.60 Section 610.60 Food...

  17. 47 CFR 15.19 - Labelling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...), and the resulting product is not separately tested: ER09DE03.001 (2) Label text and information should... label on these products shall be permanently affixed to the product and shall be readily visible to the... label shall be located in a conspicuous location on the device and shall contain the...

  18. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  19. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    .... Each label shall be identified as to: (1) Name and product code number as it appears on the product... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Label records. 116.3 Section 116.3..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label...

  20. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    .... Each label shall be identified as to: (1) Name and product code number as it appears on the product... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Label records. 116.3 Section 116.3..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label...

  1. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container of concentrate will have affixed thereto, before transfer, a label identifying the product and... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Label. 18.55 Section...

  2. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  3. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container of concentrate will have affixed thereto, before transfer, a label identifying the product and... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Label. 18.55 Section...

  4. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... following items shall appear on the label affixed to each container of a product capable of bearing a full label: (1) The proper name of the product; (2) The name, address, and license number of manufacturer; (3... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Container label. 610.60 Section 610.60 Food...

  5. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    .... Each label shall be identified as to: (1) Name and product code number as it appears on the product... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Label records. 116.3 Section 116.3..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label...

  6. 40 CFR 763.171 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... cannot be removed without defacing or destroying them. Product labels shall appear as in paragraph (d)(2... packaging, the label must be attached to the innermost layer adjacent to the product. If the innermost layer... product's innermost layer of product wrapping or packaging, or a label must be attached to the next...

  7. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  8. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Device labeling. 820.120 Section 820.120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each...

  9. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Device labeling. 820.120 Section 820.120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each...

  10. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must...

  11. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must...

  12. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... matrix. (g) The package label or package insert shall state the blood group antigens that have been... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... container label of Group O cells shall state: “FOR USE IN DETECTION OF UNEXPECTED ANTIBODIES” or “FOR USE...

  13. 40 CFR 600.301 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ECONOMY AND GREENHOUSE GAS EXHAUST EMISSIONS OF MOTOR VEHICLES Fuel Economy Labeling § 600.301 Labeling... each dealer shall maintain or cause to be maintained on each automobile: (1) A general fuel economy... vehicle for which a specific label is requested which has a combined FTP/HFET-based fuel economy value,...

  14. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Device labeling. 820.120 Section 820.120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each...

  15. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  16. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

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

  17. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

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

  18. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 3 2013-01-01 2013-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  19. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 3 2011-01-01 2011-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  20. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must...

  1. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must...

  2. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Device labeling. 820.120 Section 820.120 Food and... QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each manufacturer..., handling instructions, and any additional processing instructions. The release, including the date...

  3. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Device labeling. 820.120 Section 820.120 Food and... QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each manufacturer..., handling instructions, and any additional processing instructions. The release, including the date...

  4. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  5. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  6. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  7. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  8. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  9. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  10. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  11. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  12. 21 CFR 225.80 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Packaging and Labeling § 225.80 Labeling. (a... adhered to, will assure that the article is safe and effective for its intended purposes. (b)(1) Labels... medicated feed and includes adequate information for the safe and effective use of the medicated feed....

  13. Abdominal mass

    MedlinePlus

    Several conditions can cause an abdominal mass: Abdominal aortic aneurysm can cause a pulsating mass around the navel. ... This could be a sign of a ruptured aortic aneurysm, which is an emergency condition. Contact your health ...

  14. Abdominal mass

    MedlinePlus

    ... Several conditions can cause an abdominal mass: Abdominal aortic aneurysm can cause a pulsating mass around the navel. ... This could be a sign of a ruptured aortic aneurysm, which is an emergency condition. Contact your health ...

  15. Fluorine-18 labeling of proteins

    SciTech Connect

    Kilbourn, M.R.; Dence, C.S.; Welch, M.J.; Mathias, C.J.

    1987-04-01

    Two fluorine-18-labeled reagents, methyl 3-(/sup 18/F)fluoro-5-nitrobenzimidate and 4-(/sup 18/F)fluorophenacyl bromide, have been prepared for covalent attachment of fluorine-18 to proteins. Both reagents can be prepared in moderate yields (30-50%, EOB) in synthesis times of 50-70 min. Reaction of these reagents with proteins (human serum albumin, human fibrinogen, and human immunoglobulin A) is pH independent, protein concentration dependent, and takes 5-60 min at mild pH (8.0) and temperature (25-37 degrees C), in yields up to 95% (corrected). The /sup 18/F-labeled proteins are purified by size exclusion chromatography.

  16. Automated labeling in document images

    NASA Astrophysics Data System (ADS)

    Kim, Jongwoo; Le, Daniel X.; Thoma, George R.

    2000-12-01

    The National Library of Medicine (NLM) is developing an automated system to produce bibliographic records for its MEDLINER database. This system, named Medical Article Record System (MARS), employs document image analysis and understanding techniques and optical character recognition (OCR). This paper describes a key module in MARS called the Automated Labeling (AL) module, which labels all zones of interest (title, author, affiliation, and abstract) automatically. The AL algorithm is based on 120 rules that are derived from an analysis of journal page layouts and features extracted from OCR output. Experiments carried out on more than 11,000 articles in over 1,000 biomedical journals show the accuracy of this rule-based algorithm to exceed 96%.

  17. Isotope Labeling in Mammalian Cells

    PubMed Central

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

    2011-01-01

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

  18. 78 FR 24211 - Draft Guidance for Industry on Safety Considerations for Container Labels and Carton Labeling...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-24

    ... closure design (December 13, 2012, 77 FR 74196), and the third guidance will focus on minimizing risks... Container Labels and Carton Labeling Design To Minimize Medication Errors; Availability AGENCY: Food and... Labels and Carton Labeling Design to Minimize Medication Errors.'' The draft guidance focuses on...

  19. 40 CFR 168.65 - Pesticide export label and labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... policy. (3) No statements which appear on any of the product labels or labeling add new uses or claims or... paragraph (b) of this section which are not met by the immediate product labels. Supplemental labeling will... CFR 180.910, 180.920, 180.930, and 180.950, and the dye must not be a List 1 inert. (List 1 inerts...

  20. 40 CFR 168.65 - Pesticide export label and labeling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... policy. (3) No statements which appear on any of the product labels or labeling add new uses or claims or... paragraph (b) of this section which are not met by the immediate product labels. Supplemental labeling will... CFR 180.910, 180.920, 180.930, and 180.950, and the dye must not be a List 1 inert. (List 1 inerts...

  1. 27 CFR 19.642 - Statements required on labels under an exemption from label approval.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... meaning given, and be stated in the manner provided in 27 CFR part 5. (Sec. 201, Pub. L. 85-859, 72 Stat... labels under an exemption from label approval. 19.642 Section 19.642 Alcohol, Tobacco Products and... PLANTS Liquor Bottle and Label Requirements Bottle Label Requirements § 19.642 Statements required...

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

    PubMed Central

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

    2008-01-01

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

  3. Photolytic Labeling to Probe Molecular Interactions in Lyophilized Powders

    PubMed Central

    Iyer, Lavanya K.; Moorthy, Balakrishnan S.; Topp, Elizabeth M.

    2014-01-01

    Local side-chain interactions in lyophilized protein formulations were mapped using solid-state photolytic labeling-mass spectrometry (ssPL-MS). Photoactive amino acid analogs (PAAs) were used as probes and either added to the lyophilized matrix or incorporated within the amino acid sequence of a peptide. In the first approach, apomyoglobin was lyophilized with sucrose and varying concentrations of photo-leucine (L-2-amino-4, 4′-azipentanoic acid; pLeu). The lyophilized solid was irradiated at 365 nm to initiate photolabeling. The rate and extent of labeling were measured using ESI-HPLC-MS, with labeling reaching a plateau at ∼ 30 min, forming up to 6 labeled populations. Bottom-up MS/MS analysis was able to provide peptidelevel resolution of the location of pLeu. ssPL-MS was also able to detect differences in side-chain environment between sucrose and guanidine hydrochloride formulations. In the second approach, peptide GCG (1-8)* containing p-benzoyl-L-phenylalanine (pBpA) in the amino acid sequence was lyophilized with various excipients and irradiated. Peptide-peptide and peptide-excipient adducts were detected using MS. Top-down MS/MS on the peptide dimer provided amino acidlevel resolution regarding interactions and the cross-linking partner for pBpA in the solid state. The results show that ssPL-MS can provide high-resolution information about protein interactions in the lyophilized environment. PMID:24125175

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

    NASA Astrophysics Data System (ADS)

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

    2006-12-01

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

  5. Synthesis and characterization of the fluorescent probes for the labeling of Microthrix parvicella.

    PubMed

    Li, Songya; Fei, Xuening; Jiao, Xiumei; Lin, Dayong; Zhang, Baolian; Cao, Lingyun

    2016-03-01

    Although the fluorescent in situ hybridization (FISH) has been widely used to identify the Microthrix parvicella (M. parvicella), there are a few disadvantages and difficulties, such as complicated process, time consuming, etc. In this work, a series of fluorescent probes, which were modified by long-chain alkane with hydrophobic property and based on the property of M. parvicella utilizing long-chain fatty acids (LCFA), for the labeling of M. parvicella in bulking sludge were designed, synthesized, and characterized. The probes were characterized by ultraviolet-visible (UV-Vis) absorption spectra, fluorescence spectra, (1)H NMR spectra, and mass spectra, and the photostability and hydrophobic property of probes were investigated. All the results showed that the probes were quite stable and suitable for the fluorescent labeling. The probes had a large stoke shift of 98-137 nm, which was benefit for the fluorescent labeling. In the fluorescent labeling of M. parvicella by the synthesized probes, the probes had excellent labeling effects. By comparison of the images and the Image Pro Plus 6.0 analysis, the optimal concentration of the probes in the activated sludge sample for labeling was 0.010 mmol/L and the probe 3d had the best labeling. In addition, the effect of the duration time of probes was also investigated, and the results showed that the fluorescent intensity of probes hardly changed in a long period of time and it was suitable for labeling. PMID:26603763

  6. Accelerator mass spectrometry as a bioanalytical tool for nutritional research

    SciTech Connect

    Vogel, J.S.; Turteltaub, K.W.

    1997-09-01

    Accelerator Mass Spectrometry is a mass spectrometric method of detecting long-lived radioisotopes without regard to their decay products or half-life. The technique is normally applied to geochronology, but recently has been developed for bioanalytical tracing. AMS detects isotope concentrations to parts per quadrillion, quantifying labeled biochemicals to attomole levels in milligram- sized samples. Its advantages over non-isotopeic and stable isotope labeling methods are reviewed and examples of analytical integrity, sensitivity, specificity, and applicability are provided.

  7. Stigma of a label: educational expectations for high school students labeled with learning disabilities.

    PubMed

    Shifrer, Dara

    2013-01-01

    Poorer outcomes for youth labeled with learning disabilities (LDs) are often attributed to the student's own deficiencies or cumulative disadvantage; but the more troubling possibility is that special education placement limits rather than expands these students' opportunities. Labeling theory partially attributes the poorer outcomes of labeled persons to stigma related to labels. This study uses data on approximately 11,740 adolescents and their schools from the Education Longitudinal Survey of 2002 to determine if stigma influences teachers' and parents' educational expectations for students labeled with LDs and labeled adolescents' expectations for themselves. Supporting the predictions of labeling theory, teachers and parents are more likely to perceive disabilities in, and hold lower educational expectations for labeled adolescents than for similarly achieving and behaving adolescents not labeled with disabilities. The negative effect of being labeled with LDs on adolescents' educational expectations is partially mechanized through parents' and particularly teachers' lower expectations. PMID:24311756

  8. Use of Symbols in Labeling. Final rule.

    PubMed

    2016-06-15

    The Food and Drug Administration (FDA or the Agency) is issuing this final rule revising its medical device and certain biological product labeling regulations to explicitly allow for the optional inclusion of graphical representations of information, or symbols, in labeling (including labels) without adjacent explanatory text (referred to in this document as "stand-alone symbols") if certain requirements are met. The final rule also specifies that the use of symbols, accompanied by adjacent explanatory text continues to be permitted. FDA is also revising its prescription device labeling regulations to allow the use of the symbol statement "Rx only" or "[rx] only" in the labeling for prescription devices. PMID:27311137

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

    PubMed Central

    2015-01-01

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

  10. In Vitro Metabolic Labeling of Intestinal Microbiota for Quantitative Metaproteomics.

    PubMed

    Zhang, Xu; Ning, Zhibin; Mayne, Janice; Deeke, Shelley A; Li, Jennifer; Starr, Amanda E; Chen, Rui; Singleton, Ruth; Butcher, James; Mack, David R; Stintzi, Alain; Figeys, Daniel

    2016-06-21

    Intestinal microbiota is emerging as one of the key environmental factors influencing or causing the development of numerous human diseases. Metaproteomics can provide invaluable information on the functional activities of intestinal microbiota and on host-microbe interactions as well. However, the application of metaproteomics in human microbiota studies is still largely limited, in part due to the lack of accurate quantitative intestinal metaproteomic methods. Most current metaproteomic microbiota studies are based on label-free quantification, which may suffer from variability during the separate sample processing and mass spectrometry runs. In this study, we describe a quantitative metaproteomic strategy, using in vitro stable isotopically ((15)N) labeled microbiota as a spike-in reference, to study the intestinal metaproteomes. We showed that the human microbiota were efficiently labeled (>95% (15)N enrichment) within 3 days under in vitro conditions, and accurate light-to-heavy protein/peptide ratio measurements were obtained using a high-resolution mass spectrometer and the quantitative proteomic software tool Census. We subsequently employed our approach to study the in vitro modulating effects of fructo-oligosaccharide and five different monosaccharides on the microbiota. Our methodology improves the accuracy of quantitative intestinal metaproteomics, which would promote the application of proteomics for functional studies of intestinal microbiota. PMID:27248155

  11. IRMS detection of testosterone manipulated with 13C labeled standards in human urine by removing the labeled 13C.

    PubMed

    Wang, Jingzhu; Yang, Rui; Yang, Wenning; Liu, Xin; Xing, Yanyi; Xu, Youxuan

    2014-12-10

    Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ(13)C value). However, (13)C labeled standards can be used to control the δ(13)C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the (13)C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ(13)C values between Andro and ANAD (Δδ(13)CAndro-ANAD, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different (13)C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ(13)CAndro-ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ(13)CAndro-ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-(13)C labeled standards. PMID:25441891

  12. Dengue virus growth, purification, and fluorescent labeling.

    PubMed

    Zhang, Summer; Chan, Kuan Rong; Tan, Hwee Cheng; Ooi, Eng Eong

    2014-01-01

    The early events of the dengue virus life cycle involve virus binding, internalization, trafficking, and fusion. Fluorescently labeled viruses can be used to visualize these early processes. As dengue virus has 180 identical copies of the envelope protein attached to the membrane surface and is surrounded by a lipid membrane, amine-reactive (Alexa Fluor) or lipophilic (DiD) dyes can be used for virus labeling. These dyes are highly photostable and are ideal for studies involving cellular uptake and endosomal transport. To improve virus labeling efficiency and minimize the nonspecific labeling of nonviral proteins, virus concentration and purification precede fluorescent labeling of dengue viruses. Besides using these viruses for single-particle tracking, DiD-labeled viruses can also be used to distinguish serotype-specific from cross-neutralizing antibodies. Here the details of virus concentration, purification, virus labeling, applications, and hints of troubleshooting are described. PMID:24696327

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

    PubMed

    Allen, Doug K

    2016-02-01

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

  14. Label free redox capacitive biosensing.

    PubMed

    Fernandes, Flávio C Bedatty; Góes, Márcio S; Davis, Jason J; Bueno, Paulo R

    2013-12-15

    A surface confined redox group contributes to an interfacial charging (quantifiable by redox capacitance) that can be sensitively probed by impedance derived capacitance spectroscopy. In generating mixed molecular films comprising such redox groups, together with specific recognition elements (here antibodies), this charging signal is able to sensitively transduce the recognition and binding of specific analytes. This novel transduction method, exemplified here with C-reactive protein, an important biomarker of cardiac status and general trauma, is equally applicable to any suitably prepared interfacial combination of redox reporter and receptor. The assays are label free, ultrasensitive, highly specific and accompanied by a good linear range. PMID:23896524

  15. Tritium labeling of detonation nanodiamonds.

    PubMed

    Girard, Hugues A; El-Kharbachi, Abdelouahab; Garcia-Argote, Sébastien; Petit, Tristan; Bergonzo, Philippe; Rousseau, Bernard; Arnault, Jean-Charles

    2014-03-18

    For the first time, the radioactive labeling of detonation nanodiamonds was efficiently achieved using a tritium microwave plasma. According to our measurements, the total radioactivity reaches 9120 ± 120 μCi mg(-1), with 93% of (3)H atoms tightly bonded to the surface and up to 7% embedded into the diamond core. Such (3)H doping will ensure highly stable radiolabeled nanodiamonds, on which surface functionalization is still allowed. This breakthrough opens the way to biodistribution and pharmacokinetics studies of nanodiamonds, while this approach can be scalable to easily treat bulk quantities of nanodiamonds at low cost. PMID:24492594

  16. Hemoglobin Labeled by Radioactive Lysine

    DOE R&D Accomplishments Database

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  17. MASS SPECTROMETER

    DOEpatents

    White, F.A.

    1960-08-23

    A mass spectrometer is designed with a first adjustable magnetic field for resolving an ion beam into beams of selected masses, a second adjustable magnetic field for further resolving the ion beam from the first field into beams of selected masses, a thin foil disposed in the path of the beam between the first and second magnets to dissociate molecular ions incident thereon, an electrostatic field for further resolving the ion beam from the second field into beams of selected masses, and a detector disposed adjacent to the electrostatic field to receive the ion beam.

  18. Social determinants of diagnostic labels in depression.

    PubMed

    McPherson, Susan; Armstrong, David

    2006-01-01

    The role of diagnostic labels in medicine is usually that of labelling an illness as a means of communication. Control over labelling processes in medicine is ordinarily imposed via medical schools, textbooks, education or by diagnostic manuals. Diagnostic labels often change following new discoveries in underlying pathology such as 'consumption' being relabelled as 'TB' or 'cancer'. Sub-types of broad diagnostic labels also often emerge from such discoveries e.g. 'lung cancer' or 'throat cancer'. In mental health, underlying pathology is the subject of ongoing debate spanning ideas including the brain as a faulty organ, faulty genetics and environmental problems. With controversy over pathology comes controversy over labels and the idea that labels may be used not just for communication, but as devices of social and professional control, arising out of a social process. This study explores the codification of the diagnostic label 'depression' which emerged in the twentieth-century and has proliferated with numerous sub-types over the last 40 years. The aim is to examine its social determinants and context. Medline is used as a data source for professional label usage. A range of depression sub-type labels in professional use was identified. This exercise revealed many official and 'unofficial' terms in professional use. Citation rate plots by year were then generated for these depression sub-type labels. The rise and fall of different labels are examined in relation to social determinants and context, including publication of diagnostic manuals DSM and ICD, power shifts in psychiatry, the discovery of psychiatric drugs and the shift from inpatient to community care. Exploring the changing use of official and unofficial labels over time in this way provides a novel historical perspective on the concept of depression in the late twentieth-century. PMID:16009477

  19. Label-free molecular imaging

    NASA Astrophysics Data System (ADS)

    Zhang, Junqi; Li, Qi; Fu, Rongxin; Wang, Tongzhou; Wang, Ruliang; Huang, Guoliang

    2014-03-01

    Optical microscopy technology has achieved great improvements in the 20th century. The detection limit has reached about twenty nanometers (with near-field optics, STED, PALM and STORM). But in the application areas such as life science, medical science, clinical treatment and especially in vivo dynamic measurement, mutual restrictions still exist between numeric aperture/magnification and working distance, fluorescent dependent, and between resolution and frame rate/field size, etc. This paper explores a hyperspectral scanning super-resolution label free molecules imaging method based on the white light interferometry. The vertical detection resolution was approximate to 1 nm which is the thickness of a single molecular layer and dynamic measuring range of thickness reaches to 10 μm. The spectrum-shifting algorithm is developed for robust restructure of images when the pixels are overlapped. Micro-biochip with protein binding and DNA amplification could be detected by using this spectral scanning super-resolution molecules imaging in label free. This method has several advantages as following: Firstly, the decoding and detecting steps are combined into one step. It makes tests faster and easier. Secondly, we used thickness-coded, minimized chips instead of a large microarray chip to carry the probes. This accelerates the interaction of the biomolecules. Thirdly, since only one kind of probes are attached to our thickness-coded, minimized chip, users can only pick out the probes they are interested in for a test without wasting unnecessary probes and chips.

  20. Label-free photoacoustic nanoscopy

    PubMed Central

    Danielli, Amos; Maslov, Konstantin; Garcia-Uribe, Alejandro; Winkler, Amy M.; Li, Chiye; Wang, Lidai; Chen, Yun; Dorn, Gerald W.; Wang, Lihong V.

    2014-01-01

    Abstract. Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules. PMID:25104412

  1. Chemical kin label in seabirds.

    PubMed

    Célérier, Aurélie; Bon, Cécile; Malapert, Aurore; Palmas, Pauline; Bonadonna, Francesco

    2011-12-23

    Chemical signals yield critical socio-ecological information in many animals, such as species, identity, social status or sex, but have been poorly investigated in birds. Recent results showed that chemical signals are used to recognize their nest and partner by some petrel seabirds whose olfactory anatomy is well developed and which possess a life-history propitious to olfactory-mediated behaviours. Here, we investigate whether blue petrels (Halobaena caerulea) produce some chemical labels potentially involved in kin recognition and inbreeding avoidance. To overcome methodological constraints of chemical analysis and field behavioural experiments, we used an indirect behavioural approach, based on mice olfactory abilities in discriminating odours. We showed that mice (i) can detect odour differences between individual petrels, (ii) perceive a high odour similarity between a chick and its parents, and (iii) perceive this similarity only before fledging but not during the nestling developmental stage. Our results confirm the existence of an individual olfactory signature in blue petrels and show for the first time, to our knowledge, that birds may exhibit an olfactory kin label, which may have strong implications for inbreeding avoidance. PMID:21525047

  2. Inulin determination for food labeling.

    PubMed

    Zuleta, A; Sambucetti, M E

    2001-10-01

    Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection. PMID:11599989

  3. Label transfer by measuring compactness.

    PubMed

    Varga, Robert; Nedevschi, Sergiu

    2013-12-01

    This paper presents a new automatic image annotation algorithm. First, we introduce a new similarity measure between images: compactness. This uses low level visual descriptors for determining the similarity between two images. Compactness shows how close test image features lie to training image feature cluster centers. The measure provides the core for a k-nearest neighbor type image annotation method. Afterward, a formalism for defining different transfer techniques is devised and several label transfer techniques are provided. The method as whole is evaluated on four image annotation benchmarks. The results on these sets validate the accuracy of the approach, which outperforms many state-of-the-art annotation methods. The method presented here requires a simple training process, efficiently combines different feature types and performs better than complex learning algorithms, even in this incipient form. The main contributions of this paper are the usage of compactness as a similarity measure that enables efficient low level feature comparison and an annotation algorithm based on label transfer. PMID:23955754

  4. Inertial Mass

    ERIC Educational Resources Information Center

    King, Kenneth P.

    2007-01-01

    The inertial balance is one device that can help students to quantify the quality of inertia--a body's resistance to a change in movement--in more generally understood terms of mass. In this hands-on activity, students use the inertial balance to develop a more quantitative idea of what mass means in an inertial sense. The activity also helps…

  5. Neutron-encoded mass signatures for multiplexed proteome quantification.

    PubMed

    Hebert, Alexander S; Merrill, Anna E; Bailey, Derek J; Still, Amelia J; Westphall, Michael S; Strieter, Eric R; Pagliarini, David J; Coon, Joshua J

    2013-04-01

    We describe a protein quantification method called neutron encoding that exploits the subtle mass differences caused by nuclear binding energy variation in stable isotopes. These mass differences are synthetically encoded into amino acids and incorporated into yeast and mouse proteins via metabolic labeling. Mass spectrometry analysis with high mass resolution (>200,000) reveals the isotopologue-embedded peptide signals, permitting quantification. Neutron encoding will enable highly multiplexed proteome analysis with excellent dynamic range and accuracy. PMID:23435260

  6. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... is centered. The band at the top of the label contains the name of the fuel. This band should measure 1″ (2.54 cm) deep. Spacing of the fuel name is 1/4″ (.64 cm) from the top of the label and 3/16.... “Helvetica black” type is used throughout. All type is centered. The band at the top of the label...

  7. 75 FR 81943 - Appliance Labeling Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-29

    ...The Commission proposes changing the effective date for its new light bulb labeling requirements (published on July 19, 2010, 75 FR 41696) to January 1, 2012, to provide manufacturers with additional time to incorporate the new label on their packaging. The Commission also proposes not requiring the new label for incandescent bulbs (e.g., 75 watt bulbs) that, as of 2013, will not meet federal......

  8. 18O Labeling over a Coffee Break: A Rapid Strategy for Quantitative Proteomics

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

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

    1997-04-15

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

  10. Synthesis of ¹⁸O-labeled RNA for application to kinetic studies and imaging.

    PubMed

    Hamasaki, Tomohiro; Matsumoto, Takahiro; Sakamoto, Naoya; Shimahara, Akiko; Kato, Shiori; Yoshitake, Ayumi; Utsunomiya, Ayumi; Yurimoto, Hisayoshi; Gabazza, Esteban C; Ohgi, Tadaaki

    2013-07-01

    Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of (18)O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging (18)O atom into the phosphate group during the oxidation step of the synthetic cycle by using (18)O water as the oxygen donor. The (18)O label in the RNA was stable at pH 3-8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The (18)O/(16)O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of (18)O-labeled RNA, and this technique was used to determine the blood concentration of (18)O-labeled RNA after administration to mice. (18)O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies. PMID:23632164

  11. Radiochemically pure (1-/sup 14/C)valproic acid--a mixture of labeled structural isomers

    SciTech Connect

    Dickinson, R.G.; Wood, B.T.; Kluck, R.M.; Hooper, W.D.

    1986-01-01

    Ongoing studies of the disposition of valproic acid (VPA) and its glucuronide conjugate required the radiolabeled drug for greater sensitivity and tracing of oxidation metabolites. (1-/sup 14/C)VPA hereinafter called LABEL (radiochemical purity greater than 98% as determined by paper and thin layer chromatography) was purchased from Amersham International, U.K. Quantitative analysis of VPA and VPA-glucuronide in bile and urine samples from rats given VPA and tracer LABEL by our standard gas chromatographic assay showed gross discrepancies with the results obtained by liquid scintillation counting of the same extracts. Examination of the purity of LABEL was therefore undertaken. Equilibration of LABEL between various organic-aqueous solvent pairs was identical to that of authentic VPA. However, gas chromatographic-mass spectrometric analysis of the trimethylsilyl derivative of LABEL revealed it to be a mixture of labeled 2-methylheptanoic acid (approximately 60%), 2-ethylhexanoic acid (approximately 30%), and 2-propylpentanoic acid (i.e., VPA, 5-10%). The origin of the isomers of VPA in LABEL was logically traced to the synthetic procedure--coupling of the Grignard reagent of (an isomeric mixture of 2-, 3-, and 4-) chloroheptane(s) with (/sup 14/C)carbon dioxide. This result highlights the inadequacy of the quality control procedures used and reinforces the necessity for caution in accepting the quoted purity of radiolabeled drugs.

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

    PubMed

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

    2016-06-21

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

  13. Quantitative proteomics by amino acid labeling identifies novel NHR-49 regulated proteins in C. elegans

    PubMed Central

    Fredens, Julius; Færgeman, Nils J.

    2012-01-01

    Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology to quantitatively examine metabolic and signaling pathways in yeast, fruit flies, plants, cell cultures and mice. However, only metabolic labeling using 15N has been applied to examine such events in the nematode Caenorhabditis elegans. We have recently shown that C. elegans can be completely labeled with heavy-labeled lysine by feeding worms on prelabeled lysine auxotroph Escherichia coli for just one generation. We applied this methodology to examine the organismal response to functional loss or RNAi mediated knock down of the transcription factor NHR-49, and found numerous proteins involved in lipid metabolism to be downregulated, which is consistent with its previously proposed function as a transcriptional regulator of fatty acid metabolism. The combined use of quantitative proteomics and selective gene knockdown by RNAi provides a powerful tool with broad implications for C. elegans biology. PMID:24058826

  14. A programmed labeling approach to image interpretation

    NASA Technical Reports Server (NTRS)

    Pore, M. D.; Abotteen, R. A. (Principal Investigator)

    1979-01-01

    Manual labeling techniques require the analyst-interpreter to use not only production film converter products but also agricultural and meteorological data and spectral aids in an integrated, judgmental fashion. To control an anticipated high variance in these techniques, a semiautomatic labeling technology was developed. The product of this technology is label identification from statistical tabulation (LIST) which operates from a discriminant basis and has the ability to measure the reliability of the label and to introduce an arbitrary bias. The development of LIST and its properties are described. Numerical results of an application are included and the evaluation of LIST is discussed.

  15. Simultaneous segmentation and statistical label fusion

    NASA Astrophysics Data System (ADS)

    Asman, Andrew J.; Landman, Bennett A.

    2012-02-01

    Labeling or segmentation of structures of interest in medical imaging plays an essential role in both clinical and scientific understanding. Two of the common techniques to obtain these labels are through either fully automated segmentation or through multi-atlas based segmentation and label fusion. Fully automated techniques often result in highly accurate segmentations but lack the robustness to be viable in many cases. On the other hand, label fusion techniques are often extremely robust, but lack the accuracy of automated algorithms for specific classes of problems. Herein, we propose to perform simultaneous automated segmentation and statistical label fusion through the reformulation of a generative model to include a linkage structure that explicitly estimates the complex global relationships between labels and intensities. These relationships are inferred from the atlas labels and intensities and applied to the target using a non-parametric approach. The novelty of this approach lies in the combination of previously exclusive techniques and attempts to combine the accuracy benefits of automated segmentation with the robustness of a multi-atlas based approach. The accuracy benefits of this simultaneous approach are assessed using a multi-label multi-atlas whole-brain segmentation experiment and the segmentation of the highly variable thyroid on computed tomography images. The results demonstrate that this technique has major benefits for certain types of problems and has the potential to provide a paradigm shift in which the lines between statistical label fusion and automated segmentation are dramatically blurred.

  16. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Register citations affecting appendix L, see the List of CFR Sections Affected, which appears in the... Part 305—Sample Labels ER29AU07.122 PROTOTYPE LABEL 1 ER29AU07.123 PROTOTYPE LABEL 2 ER29AU07.124 PROTOTYPE LABEL 3 ER29AU07.125 PROTOTYPE LABEL 4 ER29AU07.126 SAMPLE LABEL 1 ER29AU07.127 SAMPLE LABEL...

  17. F-18 labeled 3-fluorodiazepam

    SciTech Connect

    Luxen, A.; Barrio, J.R.; Bida, G.T.; Satyamurthy, N.; Phelps, M.E.

    1985-05-01

    3-Fluorodiazepam is a new and potent antianxiety agent with prolonged action. The authors found that molecular fluorine (0.5% in Ne) reacts cleanly with diazepam in freon or chloroform at room temperature to produce 3-fluorodiazepam in good yields. Successful syntheses have employed 2:1 to 5:1 molar ratios diazepam: fluorine to minimize the formation of byproducts. (/sup 18/F) 3-Fluorodiazepam, a potential candidate for PET studies, (specific activity 3-5 Ci/mmol) has been synthesized from /sup 18/F-F/sub 2/ using the same procedure, followed by column chromatographic purification (Silicagel, dichloromethane: ethyl acetate, 5:1) with a radiochemical yield of 12-20% (50% maximum) and a chemical and radiochemical purity >99% as judged by reversed-phase high pressure liquid chromatography analysis (Ultrasyl octyl column, 10 ..mu.. m, 4.6 x 250 mm i.d., 60% MeOH 40% water; flow rate, 1.0 ml/min; retention time for (/sup 18/F) fluorodiazepam, 11.4 min; for diazepam, 13.5 min; radioactivity and ultraviolet detectors). Lower radiochemical yields (5-7%), and significant formation of by-products were observed when (/sup 18/F)acetylhypofluorite, prepared in the gasphase, was used as the reagent. Readily accessible routes to /sup 18/F-labeled benzodiazepines of higher specific activity were also investigated. Approaches to the synthesis of high specific activity (>200 Ci/mmol) (/sup 18/F)3-fluorodiazepam involve nucleophilic displacement at carbon-3 (e.g. from 3-chlorodiazepam) with (/sup 18/F)fluoride ion. The results presented here demonstrate the synthetic accessibility of /sup 18/F-labeled benzodiazepines for application in neurotransmitter ligand studies with PET.

  18. Labeled nucleotide phosphate (NP) probes

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  19. Amine-reactive neutron-encoded labels for highly plexed proteomic quantitation.

    PubMed

    Hebert, Alexander S; Merrill, Anna E; Stefely, Jonathan A; Bailey, Derek J; Wenger, Craig D; Westphall, Michael S; Pagliarini, David J; Coon, Joshua J

    2013-11-01

    We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between (13)C and (15)N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass difference-relative to its nearest neighbor-so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration. PMID:23882030

  20. Amine-reactive Neutron-encoded Labels for Highly Plexed Proteomic Quantitation*

    PubMed Central

    Hebert, Alexander S.; Merrill, Anna E.; Stefely, Jonathan A.; Bailey, Derek J.; Wenger, Craig D.; Westphall, Michael S.; Pagliarini, David J.; Coon, Joshua J.

    2013-01-01

    We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between 13C and 15N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass difference—relative to its nearest neighbor—so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration. PMID:23882030

  1. Mass Deacidification.

    ERIC Educational Resources Information Center

    Harris, Carolyn

    1979-01-01

    Reviews methods being developed for mass deacidification of books to prevent deterioration of paper. The use of diethyl zinc, liquified gas, and morpholine, and the advantages, disadvantages, and cost of each are considered. A 26-item bibliography is included. (JD)

  2. The reappropriation of stigmatizing labels: the reciprocal relationship between power and self-labeling.

    PubMed

    Galinsky, Adam D; Wang, Cynthia S; Whitson, Jennifer A; Anicich, Eric M; Hugenberg, Kurt; Bodenhausen, Galen V

    2013-10-01

    We present a theoretical model of reappropriation--taking possession of a slur previously used exclusively by dominant groups to reinforce another group's lesser status. Ten experiments tested this model and established a reciprocal relationship between power and self-labeling with a derogatory group term. We first investigated precursors to self-labeling: Group, but not individual, power increased participants' willingness to label themselves with a derogatory term for their group. We then examined the consequences of such self-labeling for both the self and observers. Self-labelers felt more powerful after self-labeling, and observers perceived them and their group as more powerful. Finally, these labels were evaluated less negatively after self-labeling, and this attenuation of stigma was mediated by perceived power. These effects occurred only for derogatory terms (e.g., queer, bitch), and not for descriptive (e.g., woman) or majority-group (e.g., straight) labels. These results suggest that self-labeling with a derogatory label can weaken the label's stigmatizing force. PMID:23955354

  3. To Label or Not to Label: The Special Education Question for African Americans

    ERIC Educational Resources Information Center

    Gold, Moniqueka E.; Richards, Heraldo

    2012-01-01

    Over the years, the benefits of categorically identifying and labeling students with disabilities have been debated on many grounds, particularly when it comes to labeling African-American children who many argue are over-labeled or disproportionately represented in selected categories such as learning disabilities. In this article, the authors…

  4. 9 CFR 112.2 - Final container label, carton label, and enclosure.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... product and the number of doses in each final container shall be stated on each carton label for all... product is recommended specifically for animals, and not for humans. (e) When label requirements of a... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Final container label, carton...

  5. 9 CFR 112.2 - Final container label, carton label, and enclosure.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... product and the number of doses in each final container shall be stated on each carton label for all... product is recommended specifically for animals, and not for humans. (e) When label requirements of a... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Final container label, carton...

  6. 9 CFR 112.2 - Final container label, carton label, and enclosure.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... product and the number of doses in each final container shall be stated on each carton label for all... product is recommended specifically for animals, and not for humans. (e) When label requirements of a... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Final container label, carton...

  7. 9 CFR 112.2 - Final container label, carton label, and enclosure.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... product and the number of doses in each final container shall be stated on each carton label for all... product is recommended specifically for animals, and not for humans. (e) When label requirements of a... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Final container label, carton...

  8. 40 CFR 60.536 - Permanent label, temporary label, and owner's manual.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Performance for New Residential Wood Heaters § 60.536 Permanent label, temporary label, and owner's manual. (a... section. (2) Except for wood heaters subject to § 60.530 (e), (f), or (g), the permanent label shall... material expected to last the lifetime of the wood heater, (iv) Present required information in a manner...

  9. 40 CFR 60.536 - Permanent label, temporary label, and owner's manual.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Performance for New Residential Wood Heaters § 60.536 Permanent label, temporary label, and owner's manual. (a... section. (2) Except for wood heaters subject to § 60.530 (e), (f), or (g), the permanent label shall... material expected to last the lifetime of the wood heater, (iv) Present required information in a manner...

  10. Portion Size Labeling and Intended Soft Drink Consumption: The Impact of Labeling Format and Size Portfolio

    ERIC Educational Resources Information Center

    Vermeer, Willemijn M.; Steenhuis, Ingrid H. M.; Leeuwis, Franca H.; Bos, Arjan E. R.; de Boer, Michiel; Seidell, Jacob C.

    2010-01-01

    Objective: To assess what portion size labeling "format" is most promising in helping consumers selecting appropriate soft drink sizes, and whether labeling impact depends on the size portfolio. Methods: An experimental study was conducted in fast-food restaurants in which 2 labeling formats (ie, reference portion size and small/medium/large…

  11. 9 CFR 112.2 - Final container label, carton label, and enclosure.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Final container label, carton label, and enclosure. 112.2 Section 112.2 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING § 112.2 Final...

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

    PubMed

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

    2016-06-30

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

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

    DOEpatents

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

    2014-12-09

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

  14. 21 CFR 640.70 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Source Plasma § 640.70 Labeling. Link to an amendment published... information shall appear on the label affixed to each container of Source Plasma: (1) The proper name of the... shall follow the proper name in the same size and type of print as the proper name. If the Source...

  15. 21 CFR 211.125 - Labeling issuance.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Labeling issuance. 211.125 Section 211.125 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR FINISHED PHARMACEUTICALS Packaging and Labeling...

  16. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  17. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  18. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  19. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  20. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  1. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Labeling. 660.28 Section 660.28 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube...

  2. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 20 2011-07-01 2011-07-01 false Labeling. 94.212 Section 94.212 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General requirements. (1) Each new engine...

  3. 10 CFR 20.1904 - Labeling containers.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Labeling containers. 20.1904 Section 20.1904 Energy....1904 Labeling containers. (a) The licensee shall ensure that each container of licensed material bears... handling or using the containers, or working in the vicinity of the containers, to take precautions...

  4. 40 CFR 600.301 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 31 2012-07-01 2012-07-01 false Labeling requirements. 600.301 Section 600.301 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) ENERGY POLICY FUEL ECONOMY AND GREENHOUSE GAS EXHAUST EMISSIONS OF MOTOR VEHICLES Fuel Economy Labeling § 600.301...

  5. 40 CFR 600.301 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 30 2014-07-01 2014-07-01 false Labeling requirements. 600.301 Section 600.301 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) ENERGY POLICY FUEL ECONOMY AND GREENHOUSE GAS EXHAUST EMISSIONS OF MOTOR VEHICLES Fuel Economy Labeling § 600.301...

  6. 30 CFR 74.15 - Approval labels.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Approval labels. 74.15 Section 74.15 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL MINE SAFETY AND HEALTH COAL MINE DUST SAMPLING DEVICES General Requirements for All Devices § 74.15 Approval labels. (a) Certificate...

  7. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 21 2012-07-01 2012-07-01 false Labeling. 94.212 Section 94.212 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General requirements. (1) Each new engine...

  8. The anatomy of a laser label

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Laser labeling of fruits and vegetables is an efficient alternative to adhesive tags. The advantages of this system are numerous. In general the label consists of alphanumerical characters formed by laser generated pinhole depressions that penetrate the produce’s surface creating visible markings. H...

  9. 40 CFR 204.55-4 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... STANDARDS FOR CONSTRUCTION EQUIPMENT Portable Air Compressors § 204.55-4 Labeling. (a)(1) The manufacturer of any compressor subject to the standards prescribed in § 204.52 shall, at the time of manufacture... information hereinafter provided, to all such compressors to be distributed in commerce. (2) The label...

  10. Linguistic Labels: Conceptual Markers or Object Features?

    ERIC Educational Resources Information Center

    Sloutsky, Vladimir M.; Fisher, Anna V.

    2012-01-01

    Linguistic labels affect inductive generalization; however, the mechanism underlying these effects remains unclear. According to one similarity-based model, SINC (similarity, induction, naming, and categorization), early in development labels are features of objects contributing to the overall similarity of compared entities, with early induction…

  11. 49 CFR 172.407 - Label specifications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... appendix A to this part; or (B) For labels printed on packaging surfaces, specified in table 3 in appendix... line outer border to meet the requirements of § 172.406(d) of this subpart. (c) Size. (1) Each diamond (square-on-point) label prescribed in this subpart must be at least 100 mm (3.9 inches) on each side...

  12. 42 CFR 84.257 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Labeling requirements. (a) A warning shall be placed on the label of each gas mask, chemical-cartridge... performance of any gas mask, chemical-cartridge respirator, or powered air-purifying respirator approved under... this subpart shall be specified as follows: Chemical-cartridge respirator 1 hour. Gas mask 4...

  13. 42 CFR 84.257 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Labeling requirements. (a) A warning shall be placed on the label of each gas mask, chemical-cartridge... performance of any gas mask, chemical-cartridge respirator, or powered air-purifying respirator approved under... this subpart shall be specified as follows: Chemical-cartridge respirator 1 hour. Gas mask 4...

  14. 42 CFR 84.257 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Labeling requirements. (a) A warning shall be placed on the label of each gas mask, chemical-cartridge... performance of any gas mask, chemical-cartridge respirator, or powered air-purifying respirator approved under... this subpart shall be specified as follows: Chemical-cartridge respirator 1 hour. Gas mask 4...

  15. 19 CFR 12.18 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Labels. 12.18 Section 12.18 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY SPECIAL CLASSES OF MERCHANDISE Viruses, Serums, and Toxins for Treatment of Domestic Animals § 12.18 Labels. Each separate container of such virus, serum,...

  16. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 2 2013-01-01 2013-01-01 false Label comment. 1500.128 Section 1500.128 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment....

  17. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 2 2012-01-01 2012-01-01 false Label comment. 1500.128 Section 1500.128 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment....

  18. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Label comment. 1500.128 Section 1500.128 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment....

  19. 9 CFR 112.3 - Diluent labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... labels. Each final container of diluent, other than a liquid biological product, packaged with desiccated biological products shall bear a label that includes the following: (a) The name—Sterile Diluent. (b) True name of the biological product with which the diluent is packaged, except that when the firm...

  20. 9 CFR 112.3 - Diluent labels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... labels. Each final container of diluent, other than a liquid biological product, packaged with desiccated biological products shall bear a label that includes the following: (a) The name—Sterile Diluent. (b) True name of the biological product with which the diluent is packaged, except that when the firm...

  1. 9 CFR 112.3 - Diluent labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... labels. Each final container of diluent, other than a liquid biological product, packaged with desiccated biological products shall bear a label that includes the following: (a) The name—Sterile Diluent. (b) True name of the biological product with which the diluent is packaged, except that when the firm...

  2. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Label comment. 1500.128 Section 1500.128 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment....

  3. 9 CFR 112.3 - Diluent labels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... labels. Each final container of diluent, other than a liquid biological product, packaged with desiccated biological products shall bear a label that includes the following: (a) The name—Sterile Diluent. (b) True name of the biological product with which the diluent is packaged, except that when the firm...

  4. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 2 2014-01-01 2014-01-01 false Label comment. 1500.128 Section 1500.128 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment....

  5. 9 CFR 112.3 - Diluent labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... labels. Each final container of diluent, other than a liquid biological product, packaged with desiccated biological products shall bear a label that includes the following: (a) The name—Sterile Diluent. (b) True name of the biological product with which the diluent is packaged, except that when the firm...

  6. 76 FR 13550 - Fur Products Labeling Act

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-14

    ...In December 2010, Congress passed the Truth in Fur Labeling Act (TFLA), which amends the Fur Products Labeling Act (Fur Act) by: (1) Eliminating the Commission's discretion to exempt fur products of relatively small quantity or value from disclosure requirements; and (2) providing that the Fur Act will not apply to certain fur products obtained through trapping or hunting and sold in face to......

  7. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... single package, only one package insert per package is required. (d) Names of antibodies. Blood group... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be...

  8. 46 CFR 147.30 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Labeling. 147.30 Section 147.30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) DANGEROUS CARGOES HAZARDOUS SHIPS' STORES General Provisions § 147.30 Labeling. (a) Except as provided in paragraph (b) of this section, all immediate receptacles, containers, or packages containing...

  9. 46 CFR 147.30 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Labeling. 147.30 Section 147.30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) DANGEROUS CARGOES HAZARDOUS SHIPS' STORES General Provisions § 147.30 Labeling. (a) Except as provided in paragraph (b) of this section, all immediate receptacles, containers, or packages containing...

  10. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or...

  11. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or...

  12. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or...

  13. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or...

  14. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or...

  15. Influence of Food Labels on Adolescent Diet.

    ERIC Educational Resources Information Center

    Misra, Ranjita

    2002-01-01

    Provides information on food nutrition labels and discusses the benefits of adolescents' using them to plan healthy diets. Suggests that teachers and educators should encourage appropriate label reading education for adolescents to promote healthy eating practices. Provides definitions of nutrient content claims. (SG)

  16. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... contents of the label that you wish to use, and the reasons that you want to use it. (3) Electric vehicle... electric vehicle fuel dispensing systems. All type should be set in upper case (all caps) “Helvetica Black... alternative vehicle fuel (other than electricity) labels with disclosure of principal component only....

  17. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... contents of the label that you wish to use, and the reasons that you want to use it. (3) Electric vehicle... electric vehicle fuel dispensing systems. All type should be set in upper case (all caps) “Helvetica Black... alternative vehicle fuel (other than electricity) labels with disclosure of principal component only....

  18. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... contents of the label that you wish to use, and the reasons that you want to use it. (3) Electric vehicle... electric vehicle fuel dispensing systems. All type should be set in upper case (all caps) “Helvetica Black... alternative vehicle fuel (other than electricity) labels with disclosure of principal component only....

  19. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... contents of the label that you wish to use, and the reasons that you want to use it. (3) Electric vehicle... electric vehicle fuel dispensing systems. All type should be set in upper case (all caps) “Helvetica Black... alternative vehicle fuel (other than electricity) labels with disclosure of principal component only....

  20. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Labeling. 94.212 Section 94.212 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General requirements. (1) Each new engine...