Carbon and hydrogen stable isotope fractionation during aerobic bacterial degradation of aromatic hydrocarbons

13C/(12)C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.     

Determination of cytokinins by mass spectrometry based on stable isotope dilution.

A method for quantitative determination of individual cytokinin species has been developed, based on gas chromatography-mass spectrometry and selected ion monitoring. Deuterated internal standards were prepared for analysis of N⁶-isopentenyladenosine, N⁶-isopentenyl-2-methylthioadenosine, zeatin riboside, and 2-methylthiozeatin riboside and were tested over the range of 1 to 20 ng of endogenous cytokinin per injection, relative to 100 ng of labeled standard. An isolation procedure for extracts of cabbage hearts as a model plant source has been developed that gives maximum recovery and minimum interference for gas chromatographic-mass spectrometric measurements. The present method differs from the commonly used bioassay by its selectivity for individual cytokinin components and shortened analysis time, including extractions, of 3 days vs several weeks.     

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

Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using in-gel stable isotope labeling. In this protocol protein extracts from any source treated under two experimental conditions are resolved in two separate lanes by gel electrophoresis. Parallel gel regions of interest are reacted separately with either light or heavy isotope-labeled reagents, and the gel slices are then combined and digested with proteases. The resulting peptides are then analyzed by liquid chromatography/mass spectrometry (LC/MS) to determine relative abundance of light- and heavy-isotope lysine-containing peptide pairs and analyzed by LC/MS/MS for identification of sequence and modifications. This protocol should take approximately 24-26 h to complete, including the incubation time for proteolytic digestion. Additional time will be needed for data analysis and interpretation.     

Automated comparative proteomics based on multiplex tandem mass spectrometry and stable isotope labeling

Comparative proteomic approaches using isotopic labeling and MS have become increasingly popular. Conventionally quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was metabolically labeled with [13C6]lysine and [15N4]arginine; the other set was unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity, and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a 10-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated, and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG108 cells.     

Advances in quantitative proteomics via stable isotope tagging and mass spectrometry

The high-throughput identification and accurate quantification of proteins are essential components of proteomic strategies for studying cellular functions and processes. Techniques that are largely based on stable isotope protein or peptide labeling and automated tandem mass spectrometry are increasingly being applied in quantitative proteomic studies. Over the past year, significant progress has been made toward improving and diversifying these technologies with respect to the methods for stable isotope labeling, process automation and data processing and analysis. Advances in stable isotope protein labeling and recent biological studies that used stable isotope based quantitative proteomics techniques are reviewed.     

Carbon isotope fractionation in plants

Plants with the C₃, C₄, and crassulacean acid metabolism (CAM) photosynthetic pathways show characteristically different discriminations against ¹³C during photosynthesis. For each photosynthetic type, no more than slight variations are observed within or among species. CAM plants show large variations in isotope fractionation with temperature, but other plants do not. Different plant organs, subcellular fractions and metabolises can show widely varying isotopic compositions. The isotopic composition of respired carbon is often different from that of plant carbon, but it is not currently possible to describe this effect in detail. The principal components which will affect the overall isotope discrimination during photosynthesis are diffusion of CO₂, interconversion of CO₂ and HCO^?₃, incorporation of CO₂ by phosphoenolpyruvate carboxylase or ribulose bisphosphate carboxylase, and respiration. Theisotope fractionations associated with these processes are summarized. Mathematical models are presented which permit prediction of the overall isotope discrimination in terms of these components. These models also permit a correlation of isotope fractionations with internal CO₂ concentrations. Analysis of existing data in terms of these models reveals that CO₂ incorporation in C₃ plants is limited principally by ribulose bisphosphate carboxylase, but CO₂ diffusion also contributes. In C₄ plants, carbon fixation is principally limited by the rate of CO₂ diffusion into the leaf. There is probably a small fractionation in C₄ plants due to ribulose bisphosphate carboxylase.     

Interspecific and nutrient-dependent variations in stable isotope fractionation: experimental studies simulating pelagic multitrophic systems

Stable isotope signatures of primary producers display high inter- and intraspecific variation. This is assigned to species-specific differences in isotope fractionation and variable abiotic conditions, e.g., temperature, and nutrient and light availability. As consumers reflect the isotopic signature of their food source, such variations have direct impacts on the ecological interpretation of stable isotope data. To elucidate the variability of isotope fractionation at the primary producer level and the transfer of the signal through food webs, we used a standardised marine tri-trophic system in which the primary producers were manipulated while the two consumer levels were kept constant. These manipulations were (1) different algal species grown under identical conditions to address interspecific variability and (2) a single algal species cultivated under different nutrient regimes to address nutrient-dependent variability. Our experiments resulted in strong interspecific variation between different algal species (Thalassiosira weissflogii, Dunaliella salina, and Rhodomonas salina) and nutrient-dependent shifts in stable isotope signatures in response to nutrient limitation of R. salina. The trophic enrichment in ¹⁵N and ¹³C of primary and secondary consumers (nauplii of Acartia tonsa and larval herring) showed strong deviations from the postulated degree of 1.0‰ enrichment in δ¹³C and 3.4‰ enrichment in δ¹⁵N. Surprisingly, nauplii of A. tonsa tended to keep “isotopic homeostasis” in terms of δ¹⁵N, a pattern not described in the literature so far. Our results suggest that the diets’ nutritional composition and food quality as well as the stoichiometric needs of consumers significantly affect the degree of trophic enrichment and that these mechanisms must be considered in ecological studies, especially when lower trophic levels, where variability is highest, are concerned.     

Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling

Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.     

Determination of isotope fractionation factors and quantification of carbon flow by stable carbon isotope signatures in a methanogenic rice root model system

Methanogenic processes can be quantified by stable carbon isotopes, if necessary modeling parameters, especially fractionation factors, are known. Anoxically incubated rice roots are a model system with a dynamic microbial community and thus suitable to investigate principal geochemical processes in anoxic natural systems. Here we applied an inhibitor of acetoclastic methanogenesis (methyl fluoride), calculated the thermodynamics of the involved processes, and analyzed the carbon stable isotope signatures of CO₂, CH₄, propionate, acetate and the methyl carbon of acetate to characterize the carbon flow during anaerobic degradation of rice roots to the final products CO₂ and CH₄. Methyl fluoride inhibited acetoclastic methanogenesis and thus allowed to quantify the fractionation factor of CH₄ production from H₂/CO₂. Since our model system was not affected by H₂ gradients, the fractionation factor could alternatively be determined from the Gibbs free energies of hydrogenotrophic methanogenesis. The fractionation factor of acetoclastic methanogenesis was also experimentally determined. The data were used for successfully modeling the carbon flow. The model results were in agreement with the measured process data, but were sensitive to even small changes in the fractionation factor of hydrogenotrophic methanogenesis. Our study demonstrates that stable carbon isotope signatures are a proper tool to quantify carbon flow, if fractionation factors are determined precisely.     

Influence of pluronic F68 on oxygen mass transfer

Pluronic F68 is one of the most used shear protecting additives in cell culture cultivations. It is well known from literature that such surface‐active surfactants lower the surface tension at the gas‐liquid interface, which influences the mass transfer. In this study, the effect of Pluronic F68 on oxygen mass transfer in aqueous solutions was examined. Therefore, the gassing in/gassing out method and bubble size measurements were used. At low concentrations of 0.02 g/L, a 50% reduction on mass transfer was observed for all tested spargers and working conditions. An explanation of the observed effects by means of Higbie's penetration or Dankwerts surface renewal theory was applied. It could be demonstrated that the suppressed movement of the bubble surface layer is the main cause for the significant drop down of the k_La‐values. For Pluronic F68 concentrations above 0.1 g/L, it was observed that it comes to changes in bubble appearance and bubble size strongly dependent on the sparger type. By using the bubble size measurement data, it could be shown that only small changes in mass transfer coefficient (k_L) take place above the critical micelle concentration. Further changes on overall mass transfer at higher Pluronic F68 concentrations are mainly based on increasing of gas holdup and, more importantly, by increasing of the surface area available for mass transfer. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1278–1288, 2013     

Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling

Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.     

Calcium isotope fractionation in alpine plants

In order to develop Ca isotopes as a tracer for biogeochemical Ca cycling in terrestrial environments and for Ca utilisation in plants, stable calcium isotope ratios were measured in various species of alpine plants, including woody species, grasses and herbs. Analysis of plant parts (root, stem, leaf and flower samples) provided information on Ca isotope fractionation within plants and seasonal sampling of leaves revealed temporal variation in leaf Ca isotopic composition. There was significant Ca isotope fractionation between soil and root tissue $\Updelta^{44/42}\hbox{Ca}_{\rm root-soil} \approx -0.40\,\permille$ in all investigated species, whereas Ca isotope fractionation between roots and leaves was species dependent. Samples of leaf tissue collected throughout the growing season also highlighted species differences: Ca isotope ratios increased with leaf age in woody species but remained constant in herbs and grasses. The Ca isotope fractionation between roots and soils can be explained by a preferential binding of light Ca isotopes to root adsorption sites. The observed differences in whole plant Ca isotopic compositions both within and between species may be attributed to several potential factors including root cation exchange capacity, the presence of a woody stem, the presence of Ca oxalate, and the levels of mycorrhizal infection. Thus, the impact of plants on the Ca biogeochemical cycle in soils, and ultimately the Ca isotope signature of the weathering flux from terrestrial environments, will depend on the species present and the stage of vegetation succession.     

Protein-based stable isotope probing

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.     

Simultaneous determination of endogenous and stable isotope-labelled 6beta-hydroxycortisols in human urine by stable isotope dilution mass spectrometry

This study describes a capillary GC-MS method for the simultaneous determination of endogenous 6beta-hydroxycortisol (6beta-OHF) and its stable isotope-labelled analogue, 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6beta-OHF-2H(5)), in human urine. 6beta-Hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (cortisol-13C(4),(2)H(5)) was used as an analytical internal standard. The methoxime trimethylsilyl ether (MO-TMS) derivatization was employed for the GC-MS analysis of 6beta-OHF. Quantitation was carried out by selected-ion monitoring (SIM) of the characteristic fragment ion ([M-31](+.)) of the MO-TMS derivative of 6beta-OHF. The sensitivity limit of the present GC-MS-SIM method was found to be 25 pg per injection for 6beta-OHF (S/N ratio=5.6). The within-day reproducibility in the amounts of unlabelled and labelled 6beta-OHFs determined were in good agreement with the actual amounts added, the relative errors being less than 5.30%. The inter-assay RSDs were less than 4.95% for unlabelled and labelled 6beta-OHFs.     

Influence of impeller type on mass transfer in fermentation vessels

Radial flow Rushton impellers were compared qualitatively with axial flow hydrofoil impellers (Maxflo T and A315) at the pilot scale. Six types of impellers were compared for qualitative differences in mass transfer. Measurements were conducted using three model systems: water, glycerol and Melojel (soluble starch). Power measurements were obtained using watt transducers, which although limited in accuracy and prone to interferences, were able to provide useful qualitative monitoring results. While there was little effect of impeller type on mass transfer as measured by the rapid pressure increase technique, significant qualitative differences were observed using the rapid temperature increase technique specifically for the Melojel and glycerol model systems. The Miller correlation, relating gassed-to-ungassed power, was used effectively to qualitatively evaluate the power drop upon gassing for both the model systems and a Streptomyces fermentation for the various impeller types. A high oxygen demand Streptomcyes fermentation then was conducted in fermenters possessing each type of impeller. Performance was not adequate with the A315 impellers pumping upwards and the small diameter Maxflo T impellers. Peak titers and profiles of the estimated apparent broth viscosity varied depending upon the impeller type. Mass transfer rates generally declined with higher viscosities when other fermentation operating conditions where held constant. Overall, values for OUR, k _L a, P _g /V _L and other calculated mass transfer and power input quantities for the A315 pumping upwards and undersized Maxflo T (D _T /D _I ?=?2.3) impellers were at the lower end of the range obtained for the larger Maxflo T (D _T /D _I ?=?1.8–2.0) and A315 impellers pumping downwards. Rushton impellers generally behaved qualitatively similar to hydrofoil impellers based on these calculated quantities.     

Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.     

Measurement of 4 urinary C-18 oxygenated corticosteroids by stable isotope dilution mass fragmentography

The cortisol C-18 oxidation pathway leading to the production of 18-hydroxy- and 18-oxocortisol is expressed in adenomatous primary aldosteronism and glucocorticoid remediable aldosteronism. In order to better define the significance of the pathway and its usefulness in differential diagnosis, we have developed a stable isotope dilution mass fragmentographic method for the determination of the tetrahydro metabolites of aldosterone, 18-hydroxycorticosterone and 18-oxocortisol and of unmetabolized 18-hydroxycortisol in urine. Stereochemically correct tetrahydro steroids containing 3 deuterium atoms were synthesized from the available 3-keto-4-pregnenes in 2 steps and 1,2-deuterium-labeled 18-hydroxycortisol was prepared by selective deuteration of the 1,2-double bond of a dienone precursor. Simultaneous measurement of the 4 steroids permitted a comparison of the abnormal products of the C-18 oxidation of cortisol with the normal C-18 oxidation products of corticosterone, 18-hydroxycorticosterone and aldosterone. Application of the method to the definition of the normal range is described.     

Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry

Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke, and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200 μM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5 μM), mid (5 μM), and high (100 μM) levels of 98.2, 97.3, and 101.6%, respectively. Additional assay performance metrics include intraday and interday coefficients of variance of <6.4 and <9.9%, respectively, across the range of TMAO levels. Stability studies reveal that TMAO in plasma is stable both during storage at −80 °C for 5 years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n = 349) show a range of 0.73–126 μM, median (interquartile range) levels of 3.45 (2.25–5.79) μM, and increasing values with age. The LC/MS/MS-based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic, and environmental factors on TMAO levels.