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Results for “Isotope Labeling”

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PubMed1983

Measurement of bile acid kinetics in human serum using stable isotope labeled chenodeoxycholic acid and capillary gas chromatography electron impact mass spectrometry.

A non invasive method for measurement of bile acid kinetics in serum using (24-13C)chenodeoxycholic acid has been developed. After oral administration of 50 mg (24-13C)chenodeoxycholic acid, the exponential decay of the 13C atom percent excess was measured in serum using capillary gas chromatography mass spectrometry. This required that isotope ratios were measured with high accuracy and coefficients of variation less than 1% by means of selected ion monitoring and scan averaging. The clinical applicability was tested by repeated determination of pool size, fractional turnover and synthesis rate of chenodeoxycholic acid in one healthy volunteer. This method permitted the determination of pool size, synthesis and conversion of chenodeoxycholic acid into lithocholic acid in man without the use of radioactive tracers and without repeated duodenal intubation.

Bile Acids and Salts↗
PubMed2006

The expression and refolding of isotopically labeled recombinant Matrilysin for NMR studies.

Matrilysin (MMP7) is the smallest member of matrix metalloproteinases (MMPs) family, which are collectively responsible for remodeling of connective tissue. MMP7 plays an essential role in cancer, innate immunity, and in inflammatory disorders, and has been justified as a novel drug target. Here, we report the gene synthesis, overexpression in Escherichia coli, purification and refolding of MMP7. The gene of Matrilysin was synthesized based on PCR method and overexpressed in E. coli in the form of inclusion bodies. The protein was subsequently purified and refolded to yield sufficient quantities for structural and functional studies. The purified protein was characterized by means of MALDI-TOF mass spectroscopy and dynamic light scattering (DLS) analysis. The MS data confirms the correctness of the primary sequence, while DLS experiment proves that the protein exists as a monomeric form. A significantly optimized protocol has been worked out to prepare (15)N and/or (13)C-labeled MMP7 in minimal medium with high yields for NMR studies. Under the various conditions optimized for the purification of MMP7, the yield of the purified protein is estimated to be 18-20 mg from 0.5 L of M9 minimal media. Finally, the (15)N-1H HSQC spectrum of uniformly (15)N-labeled MMP7 sample reveals that the protein is properly folded, and exists in a well-ordered structure.

Carbon Isotopes↗
PubMed1990

Mass spectrometric determination of isotopically labeled tyrosines and tryptophans in photosynthetic reaction centers of Rhodobacter sphaeroides R-26.

A synthetic medium for growing Rhodobacter sphaeroides R-26 is developed. This medium opened the way to the preparation of photosynthetic reaction centers incorporated with L-[4'-13C]tyrosine or L-[1'-15N]tryptophan. Gas chromatography combined with mass spectroscopy was used to estimate the metabolic incorporation of the labeled amino acid into the protein. Conditions were found for near-quantitative incorporation of labeled aromatic amino acids into the reaction center.

Chromatography, Gas↗
PubMed1979

Studies on drug metabolism by use of isotopes. XXIV-Determination of 3-phenylpropyl carbamate metabolites using stable isotope labelling with deuterium or carbon-13.

Metabolism of 3-phenylproply carbamate was investigated by using a stable isotope tracer technique. 3-Phenylpropanol, 3-hydroxy-3-phenylpropanol, 3-hydroxy-3-phenylpropyl carbamate, 2,3-dihydroxy-3-phenylproply carbamate, benzoic acid and hippuric acid were identified as the rat urinary metabolites. Using the dilution analysis, the amounts of metabolites in urine and faeces in rat and man were determined. In rats, 2,3-dihydroxy-3-phenylproply carbamate and 3-phenylpropanol glucuronide were excreted into the urine as the major metabolites of this drug. On the other hand, in man, the major metabolite was hippuric acid and about 30% of the administered dose was excreted as hippuric acid in the 24 h urine. The tracer technique using a singly labelled drug with carbon-13 employed in the present study provided a reliable methods for the analysis of drug metabolites and was comparable with the tracer technique using a multilabelled drug with deuterium.

1-Propanol↗
PubMed2005

Determination of sites citrullinated by peptidylarginine deiminase using 18O stable isotope labeling and mass spectrometry.

Peptidylarginine deiminase (PADI) is an enzyme which catalyzes conversion of arginine residues into citrulline residues in proteins. Citrullination is known to be related to autoimmune diseases including rheumatoid arthritis. Previous work in this laboratory succeeded in identifying citrullinated sites of human fibrinogen by mass spectrometry, but discrimination between citrullination and deamidation of asparagines and glutamine required time-consuming and labor-intensive inspection of tandem mass spectra. In this work a stable isotope is utilized to improve on a previous method for the determination of citrullinated sites by mass spectrometry. Since an oxygen atom is incorporated into the citrulline residue from H(2)O in citrullination by PADI, peptides citrullinated in 50% H(2)(18)O would show a characteristic isotope distribution different from natural abundance, and thus determination of citrullinated sites is expected to be much easier. To verify the utility of this new method, the sites of citrullination of human fibrinogen by human PADI4 were investigated using 50% H(2)(18)O. Compared with the previous method, this new method identified citrullinated sites more easily and effectively, while both the determined citrullinated sites and protein sequence coverage were unaltered.

Amino Acid Sequence↗
PubMed1993

Evaluation of stable isotope-labeled probes in the study of solvent pharmacokinetics in human subjects.

The relationship between biomarkers of exposure (such as concentrations of toxicants in blood or breath, or metabolites in urine) and toxicant dose for individuals is influenced by many person- and episode-specific factors which contribute to overall variability in biomarker level for a given dose. This variability results in imprecise biological marker-based estimates of dose for individuals. We hypothesize that pharmacokinetic data from stable-isotope (deuterated) analogs can be used with a pharmacokinetic model to account for individual-related sources of variation, leading to more precise methods of dose estimation for individuals. To establish the degree of similarity in the pharmacokinetics of unlabeled (d0-) and fully deuterated (D8-) toluene, 21 men (ages 20-45) inhaled an equal molar mixture for 2h. Washout kinetics for both compounds were followed for 4 d in alveolar air and blood. Both compounds exhibited three-phase elimination kinetics in both fluids. The third phase was not always definable for d0-toluene because of concurrent uncontrolled environmental exposures. Considering data from only the first two phases, concentrations of d0- and d8-toluene in alveolar air and blood were well correlated for all subjects, even though pharmacokinetic parameters varied among individuals by 5-9 folds. Further experiments are needed to discern whether correlations between d0- and d8-toluene for the third phase are influenced by an isotope effect; present data support use of d8-toluene as a suitable probe for d0-kinetics.

Adult↗
PubMed2004

Characterization of n-butyl acrylate centered triads in poly(n-butyl acrylate-co-carbon monoxide-co-ethylene) by isotopic labeling and two dimensional NMR.

UNLABELLED: Poly(n-butylacrylate-co-carbon monoxide-co-ethylene) (polyEBC) samples prepared from 13C-labeled monomer, n-butyl acrylate, were characterized using two dimensional (2D) pulsed field gradient (PFG) 750 MHz NMR spectroscopy. To elucidate the complex structure of the terpolymer, 2D-1H/13C-heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were conducted by selectively exciting the enhanced resonances in the spectra of two polymer samples, one polymer resulting from synthesis with 1-13C-n-butylacrylate monomer and a second polymer obtained from a synthesis with 2-13C-n-butylacrylate monomer. High-resolution 2D-NMR combined with 13C-labeling of the polymer greatly simplifies the 2D-NMR spectra, selectively enhances the weak peaks from low occurrence B-centered triad structures, and aids in their resonance assignments. In all experiments, the sample temperature was 120 degrees C, to ensure a homogeneous solution and sufficient molecular mobility. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material (1D 13C NMR spectra of the 13C-labeled and unlabeled polymers) is available in the online version of this article at http://dx.doi.org/100.1007/s00216-003-2402-3.

Acrylates↗
PubMed1988

Measurement of urinary lactic, 3-hydroxybutyric, pyruvic and acetoacetic acids in a single analysis using selected ion monitoring and stable isotope labelling techniques.

Sodium borodeuteride treatment of urine expected to have large concentrations of pyruvic and acetoacetic acids produces lactic and 3-hydroxybutyric acids labelled with deuterium. Mass fragmentographic techniques applied to the trimethylsilyated extract of the urine spiked with more extensively labelled analogues as internal standards permit the reliable determination of lactic, pyruvic, 3-hydroxybutyric and acetoacetic acids in a single analysis.

3-Hydroxybutyric Acid↗
PubMed1995

Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative [2Fe-2S]ferredoxin.

Two alternative T7 RNA promoter/polymerase systems have been employed for the heterologous expression of a plant-type [2Fe-2S]ferredoxin, Anabaena 7120 vegetative ferredoxin, in Escherichia coli at high levels (approximately 20 mg/liter of culture). One system was used when 15N-labeling the ferredoxin uniformly by growing E. coli with 15NH4Cl as the nitrogen source; the other was used in conjunction with auxotrophic host strains to enrich the protein selectively by incorporating 2H-, 13C-, and 15N-labeled amino acids. The labeled ferredoxin samples were studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Results from 1H and 2H NMR studies of samples containing [2H alpha]Cys, [2H beta 2, beta 3]Cys, [13 C beta]-Cys, and [15N]Cys have confirmed previous cysteinyl proton resonance assignments (L. Skjeldal, W. M. Westler, B.-H. Oh, A. M. Krezel, H. M. Holden, B. L. Jacobson, I. Rayment, and J. L. Markley (1991) Biochemistry 30, 7363-7368). All four 13C NMR peaks arising from the four cysteinyl beta-carbons and all four 15N NMR peaks from the four cysteinyl nitrogens were resolved in spectra of both the oxidized and reduced ferredoxins. The nitrogen resonance of Cys46, which is located in a unique (Ala-Cys) dipeptide, was assigned by detection of 13Ci-15Ni+1 coupling in a ferredoxin sample with incorporated [13C']Ala and [15N]Cys. The nitrogen signal of Cys 41 was assigned tentatively on the basis of its chemical shift and T1 relaxation time. The cysteinyl beta-carbon resonances in the reduced state have been assigned to individual residues on the basis of correlations with their (previously assigned) beta-protons. The beta-carbons resonance from Cys46 in the oxidized state has been assigned by its correlation with the corresponding resonance in the reduced state; this was accomplished by following the progressive air oxidation of a protein sample reduced by dithionite in the presence of methyl viologen. The spin-lattice relaxation times of the beta-carbons of the two cysteines coordinated to Fe)III) were similar in the oxidized and reduced states. This suggests that the antiferromagnetic coupling present in the reduced cluster has little influence on the electronic relaxation time of the Fe(III). Studies of the temperature dependence of the 1H, 13C, and 15N signals of the cysteinyl ligands to the [2Fe-2S] cluster show that the slope of the temperature dependence (delta delta/delta T-1) can be different for different atom types within a given residue. For example, in the reduced ferredoxin, although delta delta/delta T-1 is positive for Cys49 1H beta 2 and 1H beta 3, it is negative for Cys49 13C beta. Although delta delta/delta T-1 is negative for protons of cysteines ligated to Fe(II) and positive for protons of cysteines ligated to Fe(III), it is positive for all the cysteinyl nitrogens.(ABSTRACT TRUNCATED AT 400 WORDS)

Anabaena↗
PubMed1995

Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.

Conformational changes in both free cardiac troponin C (cTnC) and in complex with a recombinant troponin I protein [cTnI(33-211), cTnI(33-80), or cTnI (86-211)] were observed by means of a combination of selective carbon-13 and spin labeling. The paramagnetic effect from the nitroxide spin label, MTSSL, attached to cTnC(C35S) at Cys 84 allowed measurement of the relative distances to the 13C-methyl groups of the 10 methionines of cTnC in the monomer or complex. All 10 1H-13C correlations in the heteronuclear single- and multiple-quantum coherence (HSMQC) spectrum of [13C-methyl] Met cTnC in the complex with cTnI(33-211) were previously assigned [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., & Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735]. In the presence of oxidized spin label, nine of the 10 Met methyl 1H-13C correlations of cTnC were significantly broadened in the cTnC(C35S) monomer. This suggests flexibility within the central helix, or interdomain D/E helical linker, bringing the N- and C-terminal domains in closer proximity than predicted from the crystallographic structure of TnC. In the spin-labeled cTnC(C35S). cTnI(33-211) complex only N-terminal Met methyl 1H-13C correlations of cTnC(C35S) were paramagnetically broadened beyond detection, whereas correlations for Met residues (103, 120, 137, and 157) in the C-terminal domain were not. Thus, complex formation with cTnI decreases interdomain flexibility and maintains cTnC in an extended conformation. This agrees with the recently published study suggesting that sTnC is extended when bound to sTnI [Olah, G. A., & Trewhella, J. (1994) Biochemistry 33, 12800-12806]. The recombinant N-terminal domain of cTnI, cTnI(33-80), gave similar results as observed with cTnI(33-211) when complexed with spin-labeled cTnC(C35S). However, complex formation with the C-terminal fragment, cTnI(86-211), which contains the inhibitory sequence, is insufficient to maintain cTnC extended to the amount observed with either cTnI(33-211) or cTnI(33-80); although compared to that observed in free cTnC, it does cause decreased flexibility in the interdomain linker. In the absence of the N-terminal domain of cTnI, there is a decrease in flexibility within the N-terminal domain of cTnC. Interestingly, the N-terminal domain of cTnC in the reduced spin-labeled complex with cTnI(86-211), in the presence of ascorbate, showed two distinct conformations which were not seen in the complex with cTnI(33-211).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Isotopic labeling and LC-APCI-MS quantification for investigating absorption of carotenoids and phylloquinone from kale (Brassica oleracea).

The ability to study bioavailability of nutrients from foods is an important step in determining the health impact of those nutrients. This work describes a method for studying the bioavailability of nutrients from kale (Brassica oleracea var. Acephala) by labeling the nutrients with carbon-13, feeding the kale to an adult volunteer, and analyzing plasma samples for labeled nutrients. Results showed that conditions for producing atmospheric intrinsically labeled kale had no detrimental effect on plant growth. Lutein, beta-carotene, retinol, and phylloquinone were analyzed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Analysis of plasma samples showed that labeled lutein peaked in plasma at 11 h (0.23 microM), beta-carotene peaked at 8 (0.058 microM) and 24 h (0.062 microM), retinol peaked at 24 h (0.10 microM), and phylloquinone peaked at 7 h (3.0 nM). This method of labeling kale with (13)C was successful for producing clearly defined kinetic curves for (13)C-lutein,(13)C-beta-carotene, (13)C-retinol, and (13)C-phylloquinone.

Adult↗
PubMed2005

Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides.

Stable isotope-enriched molecules are used as internal standards and as tracers of in vivo substrate metabolism. The accurate conversion of measured ratios in the mass spectrometer to mole ratios is complicated because a polyatomic molecule containing enriched atoms will result in a combinatorial distribution of isotopomers depending on the enrichment and number of "labeled" atoms. This effect could potentially cause a large error in the mole ratio measurement depending on which isotope peak or peaks were used to determine the ratio. We report a computational method that predicts isotope distributions over a range of enrichments and compares the predicted distributions to experimental peptide isotope distributions obtained by Fourier transform ion cyclotron resonance mass spectrometry. Our approach is accurate with measured enrichments within 1.5% of expected isotope distributions. The method is also precise with 4.9, 2.0, and 0.8% relative standard deviations for peptides containing 59, 79, and 99 atom % excess (15)N, respectively. The approach is automated making isotope enrichment calculations possible for thousands of peptides in a single muLC-FTICR-MS experiment.

Animals↗
PubMed1997

A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins.

Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity. Proteins fused in frame with CBP can be purified from crude E. coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992). Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications. To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n. The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E. coli. The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7(lac)O promoter. A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest. The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E. coli cells at 15-20 mg/1 culture. CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin. Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin. We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase. Hence, the purified fusion proteins can be labeled directly with [gamma-32P]ATP and used to probe protein-protein or protein-nucleic acid interactions.

Adenosine Triphosphate↗