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PubMed2005

Field-scale isotopic labeling of phospholipid fatty acids from acetate-degrading sulfate-reducing bacteria.

Isotopic labeling of biomarker molecules is a technique applied to link microbial community structure with activity. Previously, we successfully labeled phospholipid fatty acids (PLFA) of suspended nitrate-reducing bacteria in an aquifer. However, the application of the method to low energy-yielding processes such as sulfate reduction, and extension of the analysis to attached communities remained to be studied. To test the feasibility of the latter application, an anoxic test solution of 500 l of groundwater with addition of 0.5 mM Br- as a conservative tracer, 1.1 mM SO4(2-), and 2.0 mM [2-13C]acetate was injected in the transition zone of a petroleum hydrocarbon-contaminated aquifer where sulfate-reducing and methanogenic conditions prevailed. Thousand liters of test solution/groundwater mixture were extracted in a stepwise fashion after 2-46 h incubation. Computed apparent first-order rate coefficients were 0.31+/-0.04 day(-1) for acetate and 0.34+/-0.05 day(-1) for SO4(2-) consumption. The delta13C increased from -71.03 per thousand to +3352.50 per thousand in CH4 and from -16.15 per thousand to +32.13 per thousand in dissolved inorganic carbon (DIC). A mass balance suggested that 43% of the acetate-derived (13)C appeared in DIC and 57% appeared in CH4. Thus, acetate oxidation coupled to sulfate reduction and acetoclastic methanogenesis occurred simultaneously. The delta13C of PLFA increased on average by 27 per thousand in groundwater samples and 4 per thousand in sediment samples. Hence, both suspended and attached communities actively degraded acetate. The PLFA labeling patterns and fluorescent in situ hybridization (FISH) analyses of sediment and groundwater samples suggested that the main sulfate-reducing bacteria degrading the acetate were Desulfotomaculum acetoxidans and Desulfobacter sp. in groundwater, and D. acetoxidans in sediment.

Acetates↗

PubMed1974

Transfer RNA conformation in solution investigated by isotope labeling.

The incorporation of tritium into the C-8 position of purine residues in yeast tRNA(Phe) is shown to be markedly dependent on the conformation of the molecule. The completely unfolded molecule incorporates tritium at a rate commensurate to that expected for free purine nucleotides, but partially or completely folded forms incorporate proportionately less. The labeling of specific purine sites is determined by digesting the nucleic acid with a specific nuclease and analyzing each of the radioactive fragments produced. This analysis reveals that the amount of labeling of a purine in the folded form is strongly dependent upon its position in the sequence, whereas purine labeling in the unfolded form is independent of sequence position. The labeling pattern of the different bases in the folded form agrees fairly well with what is expected, based on existing solution and x-ray data on the conformation, i.e., residues involved in helical sections or apparently masked by tertiary interactions label more slowly than those on the "outside" of the molecule. Finally, folding and unfolding of specific regions of the macromolecule may be followed by the isotope labeling.

Base Sequence↗

PubMed2004

Nature of vibrational coupling in helical peptides: an isotopic labeling study.

Infrared (IR) and vibrational circular dichroism (VCD) spectra were measured for a series of isotopically ((13)C on two or more amide Cdouble bond]O) labeled, 25 residue, alpha-helical peptides of the sequence Ac-(AAAAK)(4)AAAAY-NH(2) that were also studied in the previous paper. Theoretical IR and VCD simulations were performed for correspondingly isotopically labeled Ac-A(24)-NHCH(3) constrained to an alpha-helical conformation by use of property tensor transfer from density functional theory (DFT) calculations on Ac-A(10)-NHCH(3). The simulations predicted and experiments confirmed that the vibrational coupling constants between i, i + 1 and i, i + 2 residues differ in sign, thus leading to a reversal of the (13)C VCD pattern and explaining the large shift in the (13)C amide I frequency as reported in the previous paper. The sign of the coupling constant remained consistent for larger label separation (with the exception of i, i + 4) and for more labels with uniform separation. Such effects confirm that the isotopically labeled group vibrations are essentially only coupled to each other and are effectively uncoupled from those of the unlabeled groups. This development confirms the utility of isotopic labels for site-specific structural studies with vibrational spectra. Observed spectral effects cannot be explained by considering only transition dipole coupling (TDC) between amide oscillators, particularly for smaller label separations, but the TDC and ab initio predicted couplings roughly converge at large separation.

Alanine↗

PubMed2006

Automated quantification tool for high-throughput proteomics using stable isotope labeling and LC-MSn.

LC-MSn has become a popular option for high-throughput quantitative proteomics, thanks to the availability of stable-isotope labeling reagents. However, the vast quantity of data generated from LC-MSn continues to make the postacquisition quantification analyses challenging, especially in experiments involving multiple samples per experimental condition. To facilitate data analysis, we developed a computer program, QUIL, for automated protein quantification. QUIL accounts for the dynamic nature of spectral background and subtracts this background accordingly during ion chromatogram reconstruction. For elution profile identification, QUIL minimizes the inclusion of coeluted neighbor peaks, yet tolerates imperfect peak shapes. Outlier-resistant methods have been implemented for better protein ratio estimation. The utility of QUIL was validated by quantitative analyses of a standard protein as well as complex protein mixtures, which were labeled with cICAT or 18O and analyzed using LCQ, LTQ, or FT-ICR instruments. For samples that no prior knowledge of relative protein quantities was available, Western blotting was performed for confirmation. For the standard protein, the coefficient of variation (CV) of peptide ratio estimation was 6%. For complex mixtures, the median CV for protein ratio calculations was less than 10%. Computed protein abundance ratios exhibited a relatively high degree of correlation with those obtained from Western blot analyses. Compared with a widely used commercial software tool, QUIL showed improvement in ion chromatogram construction and peak integration and significantly reduced relative errors in abundance ratio assessment.

Animals↗

PubMed1982

Differentiation of isotope-labeled myeloid leukemia cells free in the peritoneal cavity of syngeneic mice.

Ml cells were labeled in vitro with [3H]thymidine and injected into the peritoneal cavity of syngeneic mice. After several days the peritoneal cells were harvested and [3H]labeled cells were determined by autoradiography. The inoculated isotope-labeled Ml cells differentiated in the peritoneal cavity and lipopolysaccharide, an inducer of cell differentiation, significantly stimulated differentiation. These results provide direct evidence that Ml cells can be induced to differentiate in vivo under conditions in which leukemia can develop.

Animals↗

PubMed1992

Evaluation of a Salmonella-specific DNA probe by colony hybridization using non-isotopic and isotopic labeling.

A 2.3 kilobase (kb) Salmonella probe, JEO402-1, and two subfragments, F1214 (1.3 kb) and F1217 (0.8 kb), have been evaluated by colony hybridization using pure cultures of Salmonella serovars and non-salmonella bacteria. JEO402-1, and its subfragments, F1214 and F1217, hybridized to all of 156 different Salmonella serovars tested, while there was no reaction to 112 non-salmonella strains belonging to 19 genera and 37 species of Enterobacteriaceae. Together with previously published results, the JEO402-1 probe has now been shown to detect a total of 396 Salmonella strains belonging to 214 serovars of Salmonella subspecies I-VI. A total of 178 non-salmonella strains representing 23 genera and 51 species of Enterobacteriaceae have all tested negative with JEO402-1. The hybridization results obtained using a digoxigenin-labeled probe were similar to those obtained with 35S isotopic labeling when complete colony lysis was ensured.

Bacterial Typing Techniques↗

PubMed1990

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

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.

Animals↗

PubMed1980

Resonance Raman spectroscopy of chemically modified and isotopically labelled purple membranes. II. Kinetic studies.

Kinetic resonance Raman spectra of native and isotopically labelled purple membranes are compared. Using these data and the assignments of the previous paper in this sequence, we have confirmed that the Schiff base is deprotonated at times that are short in comparison to M412 evolution. In addition, by monitoring the kinetic resonance Raman spectra in 2H2O with 488.0 nm excitation we have been able to characterize in more detail the vibrational features associated with this unprotonated intermediate that precedes M412. Furthermore, the kinetic spectra of fully deuterated purple membrane in H2O have allowed us to assign the 1465 cm-1 band in these spectra to the C=C stretching frequency of BR570 and the 1512 cm-1 band to the C=C stretching frequency of M412. These spectra have also provided an indication of a Raman spectral feature associated with O640 and, finally, our kinetic spectra have provided evidence that there is a significant alteration in the rate constants for the evolution of the various intermediates when the non-exchangeable protons on the membrane are replaced by deuterons.

Bacteriorhodopsins↗

PubMed1987

Schistosoma mansoni: autoradiographic tracking studies of isotopically-labelled challenge parasites in naive and vaccinated CBA/Ca mice.

The migration of isotopically-labelled challenge parasites of Schistosoma mansoni in naive CBA/Ca mice, and CBA/Ca mice vaccinated 4 weeks previously with about 600 radiation-attenuated cercariae, has been followed by means of compressed organ autoradiography. In naive mice, only 16% of the challenge parasites failed to migrate from the skin to the lungs, whereas up to half of the individuals that succeeded in reaching the pulmonary vasculature did not move on to the liver. The tracking technique thus revealed a total loss of 58% of the challenge parasites, which correlated well with the fact that only 50% of the challenge was recovered as adult worms by retrograde perfusion of the hepatic portal system. Challenge migration in vaccinated mice initially proceeded more slowly than in naive mice, but peak numbers of foci were eventually recorded in the lungs on the same day in both groups of individuals. We did not therefore recognize a delay in parasite migration in vaccinated mice. In the present experiments, 58.5% of the challenge failed to reach the lungs of vaccinated rodents, and 25% of those parasites that did attain the pulmonary vasculature were not recruited to the liver. The tracking technique thus accounted for a total loss of 83.5% of the parasites, which again correlated well with the fact that we recovered only 22% of the challenge as adult worms at portal perfusion. The data presented here prove conclusively that the major phase of immune-dependent challenge elimination in vaccinated CBA/Ca mice occurs in the cutaneous tissues, and that only a small proportion of the parasites are lost in the lungs. These data are entirely consistent with those we have published elsewhere for the CBA/Ca mouse using a multiplicity of different techniques; they differ however, from results reported by others for the C57 Black strain of mouse. Possible reasons for these discrepancies are discussed.

Animals↗

PubMed1975

Structural analyses of triglycerides containing isotopically-labelled fatty acids.

The stereospecific analysis of triglycerides is a complex, multistep procedure and some losses inevitably occur at every stage. Such losses are particularly important when the triglycerides contain isotopically-labelled fatty acids incorporated by incubation in vitro with the relevant enzyme systems, but they can be compensated for by adding, prior to the analysis, a triglyceride containing only the same labelled odd-chain fatty acid in each position.

Carbon Radioisotopes↗

PubMed1995

Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.

For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity of labeling in in vivo expression systems. In the present study, a cell-free protein synthesis system was optimized, so that highly efficient and selective stable isotope labeling of proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymerase for transcription and also by improving the translation conditions. Thus, about 0.1 mg protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-labeling of the human c-Ha-Ras protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras protein was obtained. 1H-15N HSQC experiments confirmed that the Ras protein was efficiently labeled with high selectivity. These results indicate that this cell-free protein synthesis system is useful for NMR studies.

Amino Acids↗

PubMed2005

Determination of complex isotopomer patterns in isotopically labeled compounds by mass spectrometry.

A classic problem in analytical chemistry has been determination of individual components in a mixture without availability of the pure individual components. Measurement of the distribution of isotopomers in a labeled compound or mixture of labeled compounds is an example of this problem that is commonly encountered when stable isotopically labeled metabolites are used to determine in vivo kinetics and metabolism. We present a method that uses the measured mass spectral data of the unlabeled material to represent any and all combinations of isotopomer variations of that material and to determine abundances of these isotopomers. Although examples of the method are presented for gas chromatography/mass spectrometry, the method is applicable to any type of mass spectrometry data. The method also accounts for errors induced by mass spectrometer ionization and resolution effects. To demonstrate this method, we determined the isotopomer distributions of samples of 13C-labeled leucine and glucose for both highly enriched isotopomers and labeled isotopomers present in low abundance against a natural isotopic abundance background. The method accurately and precisely determined isotopomer identity and abundance in the labeled materials without adding noise or error that was not inherent in the original mass spectral data. In examples shown here, isotopomer uncertainties were calculated with relative standard errors of <1% from good quality mass spectral data.

Glucose↗

PubMed1989

Binding of isotopically labeled substrates, inhibitors, and cyanide by protocatechuate 3,4-dioxygenase.

Binding of ligands to the active site Fe3+ of protocatechuate 3,4-dioxygenase is investigated using EPR-detected transferred hyperfine coupling from isotopically labeled substrates, inhibitors, and cyanide. Broadening is observed in EPR resonances from the anaerobic enzyme complex with homoprotocatechuate (3,4-dihydroxyphenylacetate), a slow substrate, enriched with 17O (I = 5/2) in either the 3-OH or the 4-OH group. This shows that this substrate binds directly to the Fe3+ and strongly suggests that an iron chelate can be formed. Cyanide is known to bind to the enzyme in at least two steps, forming first a high spin and then a low spin complex (Whittaker, J. W., and Lipscomb, J. D. (1984) J. Biol. Chem. 259, 4487-4495). Hyperfine broadening from [13C]cyanide (I = 1/2) is observed in the EPR spectra of both complexes, showing that cyanide is an Fe3+ ligand in each case. Cyanide binding is also at least biphasic in the presence of protocatechuate (PCA). The initial high spin enzyme-PCA-cyanide complex forms rapidly and exhibits a unique EPR spectrum. Broadening from PCA enriched with 17O in either the 3-OH or the 4-OH group is detected showing that PCA binds to the iron, probably as a chelate complex. In contrast, no broadening from [13C]cyanide is detected for this complex suggesting that cyanide binds at a site away from the Fe3+. Steady state kinetic measurements of cyanide inhibition of PCA turnover are consistent with two rapidly exchanging cyanide binding sites that inhibit PCA binding and which can be simultaneously occupied. Formation of the nearly irreversible, low spin enzyme-PCA-cyanide complex is competitively inhibited by PCA. Transient kinetics of the formation of this complex are second order in cyanide implying that two cyanides bind. Broadening in the EPR spectrum of this complex is detected from [13C]cyanide, but not from [17O]PCA, suggesting that PCA is displaced. This study provides the first direct evidence for chelation of the active site Fe3+ by substrates and for a small molecule binding site away from the iron in intradiol dioxygenases.

Binding Sites↗

PubMed1999

Endogenous surfactant metabolism in critically ill infants measured with stable isotope labeled fatty acids.

Little is known about endogenous surfactant metabolism in infants, because radioactive isotopes used for this purpose in animals cannot be used in humans. We developed a novel and safe method to measure the endogenous surfactant kinetics in vivo in humans by using stable isotope labeled fatty acids. We infused albumin-bound [U-13C]palmitic acid (PA) and [U-13C]linoleic acid (LLA) for 24 h in eight critically ill infants (mean+/-SD: weight: 3.7+/-1.3 kg: age: 51.3+/-61.6 d) who required mechanical ventilation. The 13C enrichment of PA and LLA in surfactant phosphatidylcholine (PC), obtained from tracheal aspirates, was measured by gas chromatography combustion interface-isotope ratio mass spectrometry. We measured a significant incorporation of both 13C-PA and 13C-LLA into surfactant PC. PC-PA and PC-LLA became enriched after 8.7+/-4.9 h (range: 3.4-17.3) and 10.0+/-7.2 h (range: 3.0-22.4), respectively; the times at maximum enrichment were 49.2+/-8.9 and 45.6+/-19.3 h, respectively. The fractional synthesis rate of surfactant PC-PA ranged from 0.4 to 3.4% per h, whereas the fractional synthesis rate of PC-LLA ranged from 0.5 to 3.8% per h. The surfactant PC-PA and PC-LLA half-lives ranged from 16.8 to 177.7 and 23.8 to 144.4 h, respectively. This method provides new data on surfactant metabolism in infants requiring mechanical ventilation. We found that synthesis of surfactant from plasma PA and LLA is a slow process and that there were marked differences in PC kinetics among infants. This variability could be related to differences in lung disease and could affect the clinical course of the respiratory failure.

Carbon Isotopes↗

PubMed1976

Isotope labeling of free and aminoacyl transfer RNA synthetase-bound transfer RNA.

The structural organization of the complex of Escherichia coli Ile-tRNA synthetase and tRNA Ile has been studied by isotope labeling of purine units in the tRNA. Free or bound tRNA is incubated in tritiated water for 5 to 15 h at 37 degrees in order to incorporate tritium into the C-8 positions of purine units. (Previous work has shown that the labeling rate of a purine is very sensitive to its microenvironment.) Under conditions where exchange-out does not occur, the nucleic acid is digested with nucleases and purines are subsequently isolated from known locations in the structure. Four purines are substantially perturbed by bound Ile-tRNA synthetase; in each case, the rate of labeling is retarded in the presence of the synthetase. The four purines occur at or near the 3' terminus and at the interface of the dihydrouridine stem and loop. These bases occur in segments of the tRNA that previous photochemical cross-linking studies have identified as important for synthetase-tRNA interactions. It appears that the effects observed on these sites are caused by their direct interaction with or shielding by the bound synthetase. In addition, two other sites, one in the anticodon and one in the amino acid acceptor-T psi C helix, appear to be perturbed (retarded labeling rates) by the bound enzyme. The data also suggest there is no significant conformational change in the tRNA upon binding to the synthetase.

Adenine Nucleotides↗

PubMed1997

Energy expenditure using isotope-labelled water (2H218O), exercise performance, skeletal muscle enzyme activities and plasma biochemical parameters in humans during 95 days of endurance exercise with inadequate energy intake.

Two men, R.F. and M.S., pulled sledges each with starting masses of 222 kg, 2300 km across Antarctica. Exercise was performed for approximately 10 h each day for 95 days. Despite an average energy intake of 21.3 MJ.day-1 both subjects lost more than 25% of body weight. Energy expenditure was measured using energy balance data (EB) and isotope-labelled water (2H218O). Isotope doses were taken on day 0 and day 50 of the expedition. During the first 50 days both methods gave reasonable agreement, giving energy expenditures of 38.3 (EB) and 35.5 (2H218O) MJ.day-1 in R.F. and 28.6 (EB) and 29.1 (2H218O) MJ.day-1 in M.S. The isotope data for days 20-30 yielded exceptional values of 44.6 MJ.day-1 in R.F. and 48.7 MJ.day-1 in M.S. Estimates of energy expenditure between day 51 and day 96 were much lower and although the methods were in agreement for R.F.-24.1 (EB) and 23.1 (2H218O) MJ. day-1, there was poor agreement for MS-26.8 (EB) and 18.8 (2H218O) MJ.day-1. However, some practical difficulties occurred during this second period and there were also problems arising from marked increases in body water that made estimates of body mass and composition change difficult to interpret. The latter problems were probably due to malnutrition, which may have also been responsible for surprising increases in urinary excretion of 2H and 18O observed in both men at around day 81. These increases may reflect the release of label incorporated into molecules other than water which do not normally freely exchange with the body water pool under the circumstances of marked malnourishment. Following the expedition, both men showed declines in maximal O2 consumption (VO2max, 53.6 to 41.2 ml O2 kg-1.min-1 in R.F., 58.1-46.0 ml O2 kg-1.min-1 in M.S.); maximal voluntary isometric force production in different muscle groups (up to 19.9% in R.F. and 55.8% in M.S.) and both cytoplasmic and mitochondrial skeletal muscle enzyme activities (up to 56% in R.F. and 63% in M.S.). Plasma samples taken during the expedition showed low glucose levels, inappropriately high insulin levels, and declines in testosterone and luteinizing hormone. Thyroxine, cholesterol, albumin and triglyceride levels remained normal.

Adult↗

PubMed1996

Stable isotope labelling and determination of ketroprofen in human plasma and urine by gas chromatography/negative ion chemical ionization mass spectrometry.

A method for the quantitative measurement of the non-steroidal anti-inflammatory drug ketoprofen in human plasma and urine is presented. The assay is based on gas chromatography/negative ion chemical ionization mass spectrometry. The preparation of stable isotope-labelled ketoprofen for use as an internal standard is described. After solvent extraction from the acidified matrix, urine samples were analysed as pentafluorobenzyl esters, whereas plasma samples required further derivatization to the hydroxylamine-trimethylsilyl derivatives. The detection limit was found to be 5 pg in both cases. The method was applied to the pharmacokinetics of ketoprofen in man after epidermal application.

Administration, Topical↗

PubMed2006

Rapid preparation of stable isotope labeled peptides that bind to target proteins by a phage library system.

We have developed a system for directly isolating foreign peptides displayed on the N-terminus of the major coat protein of bacteriophage M13. The phage particle in this system is formed as a mixture of wild type and modified coat proteins. The N-terminal segment of the modified coat protein was mutated for chemical cleavage, in order to obtain the displayed peptide from the major coat protein. Using 13C, 15N- labeled medium, we introduced stable isotopes, 13C and/or 15N, into the coat proteins. The NMR spectra for the cleaved peptides from the phage particles could be recorded within a few days after the selection of the phage clone.

Amino Acid Sequence↗