Properties of DNA from Various Local Strains of Bradyrhizobium sp. (Vigna radiata)

The buoyant density of DNA from various local strains of mung-bean nodule bacteria (Bradyrhizobium sp.) was determined by ultracentrifugation in an equilibrium CsCl gradient. The number of GC base pairs (61.1-62.7% mol %) and DNA melting point (94.3-95.0°C) were calculated from the buoyant density. DNA preparations purified by ultracentrifugation in a CsCl gradient by electrophoresis show a bimodal distribution. The DNA composition varies little (1.6 mol %). The electrophoretic distribution of local and Type-strain mung-bean nodule bacteria are identical. This proves that they belong to a single taxonomic group.     

Comparison of methods for simultaneous identification of bacterial species and determination of metabolic activity by protein‐based stable isotope probing (Protein‐SIP) experiments

We developed a concept for analysing carbon and nitrogen fluxes in microbial communities by employing protein‐based stable isotope probing (Protein‐SIP) in metabolic labelling experiments with stable isotope labelled substrates. For identification of microbial species intact protein profiling (IPP) can be used, whereas the assessment of their metabolic activity is achieved by shotgun mass mapping (SMM). Microbial cultures were grown on substrates containing ¹³C or ¹⁵N. For identification of species we tested both the IPP and the SMM approaches. Mass spectra (MALDI‐MS) were taken from mixtures of either intact proteins or peptides from tryptic digestion for generating species‐specific peak patterns. In the case of SMM, the fragmentation of peptides was additionally used to obtain sequence information for species identification. Mass spectra of peptide sequences allow calculation of the amount of ¹³C or ¹⁵N incorporation within peptides for determining metabolic activity of the specific species. The comparison of IPP and SMM revealed a higher robustness of species identification by SMM. In addition, the assessment of incorporation levels of ¹³C and ¹⁵N into peptides by SMM revealed a lower uncertainty (0.5–0.8 atom %) compared to IPP (6.4–8.9 atom %). The determination of metabolic activity and function of individual species by Protein‐SIP can help to analyse carbon and nitrogen fluxes within microbial communities. Copyright © 2009 John Wiley & Sons, Ltd.     

Technical considerations for the use of 15N-DNA stable-isotope probing for functional microbial activity in soils

Stable-isotope DNA probing is a culture-independent technique that may provide a link between function and phylogeny of active microorganisms. The technique has been used in association with 13C substrates while here we evaluate feasibility and limitations of 15N-DNA stable-isotope probing (SIP) using labelled and unlabelled pure microbial cultures or soil extracts. Our results showed that (15)N-DNA probing is feasible for cultures as well as soil samples. Limitations of 15N-DNA-SIP are (a) the need for relatively large quantities of DNA to visualise bands (although molecular resolution is much higher) and (b) 15N-DNA enrichment needed to ideally be >50 at%; however, this requirement can be lowered to approx. 40 atom% 15N with pure cultures using a modified CsCl centrifugation method (140K g for 69 h). These advances in 15N-DNA-SIP methodology open new opportunities to trace active microbial populations utilising specific N substrates in situ.     

Short-term dynamics of slurry-derived plant and microbial sugars in a temperate grassland soil as assessed by compound-specific delta13C analyses

In view of recent discussions about climate change and the anthropogenically enhanced greenhouse effect, the aim of this study was to determine the short-term carbon (C) dynamics in a grassland soil after slurry application. It is known that, depending on cultivation practices, agro-ecosystems can act either as sources or as sinks for atmospheric CO2. C3 and C4 slurries were applied, differing in their stable C isotope signature, to be able to differentiate between native (soil-inherent) and fresh (slurry-applied) C. Samples were taken from 0-2, 2-7.5 and 7.5-15 cm soil depths from 90 days before until 4 weeks after slurry application at various intervals. We carried out compound-specific stable isotope analysis (CSIA) of plant- (arabinose and xylose) and microbial-derived sugars (fucose and rhamnose). Up to 45% of the applied slurry-derived xylose was found in the 0-2 cm soil depth within 24 h after slurry application, with this figure decreasing rapidly and then increasing again towards the end of the experiment. Therefore, during the first phase of slurry incorporation, preferentially the soluble part of slurry entered the first 2 cm of soil while, after about 2 weeks, particulate slurry-derived organic matter was incorporated into the soil. The ratio between plant- and microbial-derived sugars together with delta13C values of individual sugars in the 2-7.5 cm soil depth revealed that the dissipation of sugars from the 0-2 cm soil depth was not only due to leaching, but also was caused by microbial degradation of the fresh C because slurry did not contain significant amounts of rhamnose while the delta13C values of rhamnose became progressively enriched in 13C during the experiment. Stable isotope measurements of bulk soil previously only showed significant differences between C4 and C3 plots at 0-2 cm soil depth. The CSIA of the individual sugars was much more sensitive than bulk isotope measurements, revealing significant differences between C4 and C3 plots even at the 2-7.5 cm soil depth during the first phase of the experiment. Additionally, the dynamics of slurry-derived plant and microbial sugars could be followed specifically.     

The Effect of Temperature on the Structure and Function of a Cellulose-Degrading Microbial Community

The purpose of this study was to investigate the effect of temperature on the structure and straw degradation capability of a microbial community grown from wheat straw compost. Two cellulolytic microbial communities, WDC1 and WDC2, were obtained from compost. The communities had been cultured under 50 and 60?°C by continuous enrichment, respectively. The wheat straw degradation capabilities were 45.69?% (WDC1) and 59.5?% (WDC2). By changing the culture temperatures, two new stable communities were obtained: WDC1-6N (WDC1, cultivated at 60?°C for eight generations) and WDC2-5N (WDC2, cultivated at 50?°C for eight generations). The wheat straw degradation capabilities for the new communities were 59.75 and 52.60?%, respectively. The results showed that compared to 50?°C, the wheat straw degradation capability of the communities cultured at 60?°C was stronger. Sequencing of selected denaturing gradient gel electrophoresis (DGGE) bands and analysis of DGGE profiles indicated that the WDC2 structure was significantly different from the structure of WDC1. This was so even though the two communities were enriched from the same compost. With the change of culture temperature, the community structures underwent significant transitions. Included communities were thermophilic, anaerobic bacteria, and any cellulolytic bacteria (e.g., Clostridium thermocellum) that were active and abundant at conditions under 60?°C. These results have the potential to significantly aid in the enrichment of a cellulose-degrading community from the environment and to enhance the community capability to conduct straw biotransformation.     

Capillary electrophoresis ribosomal RNA single-stranded conformation polymorphism: a new approach for characterization of low-diversity microbial communities

Capillary electrophoresis (CE) has been the principle system for nucleic acid analysis since the early 1990s due to its inherent advantages such as fast analysis time, high resolution and efficiency, minimal sample requirement, high detection sensitivity, and automation. In the past few decades, microbial community fingerprinting methods such as terminal restriction fragment length polymorphism and single-stranded conformation polymorphism (SSCP) have migrated to CE to utilize its advantages over conventional slab gel electrophoresis. Recently, a gel-based direct rRNA fingerprint method was demonstrated. Different from other existing microbial community characterization approaches, this novel approach is polymerase chain reaction free and capable of providing information on the relative abundance of rRNA from individual phylotypes in low-diversity samples. As a gel-based method, it has a long analysis time and relatively large reagent and sample requirements. Here, we addressed these limitations by transferring the RNA fingerprint approach to the CE platform. Analysis time significantly improved from 24?h to 60?min, and the use of a fluorescently labeled hybridization probe as the detection strategy decreased the sample requirement by ten-fold. The combination of fast analysis time, low sample requirement, and sensitive fluorescence detection makes CE-RNA-SSCP an appealing new approach for characterizing low-diversity microbial communities.     

Isolation of peroxisomal subpopulations from mouse liver by immune free-flow electrophoresis

Peroxisomes (PO) are a heterogeneous population of cell organelles which in mammals are most abundant in liver and kidney. Commonly, differential and density gradient centrifugation are used for their isolation which, however, give only rise to the so-called "heavy" PO with a buoyant density of 1.22-1.24 g/cm(3). Subpopulations other than the heavy PO which are also present in both of these tissues have escaped adequate purification because of their sedimentation characteristics which are close to those of other major organelles, in particular microsomes. Since the purification of these subpopulations has become an essential task in view of the putative importance of peroxisomal subpopulations in the biogenesis of this organelle, alternatives to density gradient centrifugation are required. Recently, we have introduced such a novel approach, named immune free flow electrophoresis (IFFE). IFFE combines the advantages of eletrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle, complexed with an antibody directed against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of immunoglobulin G (IgG) molecules. pH-values other than 8.0 proved to be less efficient, probably because IgG molecules only focus at pH 8.0 but are scattered at any other. Applying IFFE to heavy and light mitochondrial as well as microsomal fractions of rat liver not only regular PO (rho = 1.22-1.24 g/cm(3)) but also other subpopulations could be isolated. To substantiate the validity of this approach, we now have subfractionated mouse liver homogenates accordingly. Of the PO subpopulations collected, mainly that obtained from the heavy mitochondrial fraction differed in its composition of matrix and membrane proteins as revealed by immunoblotting. This is in line with the data reported on rat liver thus confirming the potential of IFFE in the isolation of distinct subpopulations of hepatic PO.     

Deuterium isotope effects on 13C chemical shifts of negatively charged NH…N systems

Deuterium isotope effects on ¹³C chemical shifts are investigated in anions of 1,8‐bis(4‐toluenesulphonamido)naphthalenes together with N,N‐(naphthalene‐1,8‐diyl)bis(2,2,2‐trifluoracetamide) all with bis(1,8‐dimethylamino)napthaleneH⁺ as counter ion. These compounds represent both “static” and equilibrium cases. NMR assignments of the former have been revised. The NH proton is deuteriated. The isotope effects on ¹³C chemical shifts are rather unusual in these strongly hydrogen bonded systems between a NH and a negatively charged nitrogen atom. The formal four‐bond effects are found to be negative indicating transmission via the hydrogen bond. In addition, unusual long range effects are seen. Structures, ¹H and ¹³C NMR chemical shifts and changes in nuclear shieldings upon deuteriation are calculated using density functional theory methods. Copyright © 2013 John Wiley & Sons, Ltd.     

Carbon assimilation and turnover in grassland vegetation using an in situ (13)CO(2) pulse labelling system

A mobile laboratory was developed to administer a controlled flow of (13)C labelled CO(2) at ambient concentrations ( approximately 350 ppm) in the field. The stable isotope delivery (SID) system consists of an isotope-mixing unit with flow control to a series of 12 independent labelling chambers. In-line CPU controlled infrared gas analysers allow automated measurement of chamber CO(2) concentrations and gas flow management. A preliminary experiment was established on an upland pasture located at the NERC Soil Biodiversity experimental site, Sourhope, UK, in August 1999. The objective of this investigation was to determine the magnitude of pulse-derived C incorporation into a typical upland plant community. To achieve this, the SID system was deployed to pulse-label vegetation with CO(2) enriched with (13)C (50 atom %) at ambient concentrations ( approximately 350 ppm) on two consecutive days in August 1999. Samples of headspace CO(2), shoot and root were taken on four occasions over a period of 28 days after (13)C labelling. These materials were then prepared for (13)C/(12)C ratio determination by continuous-flow/combustion/isotope ratio mass spectrometry (CF-C-IRMS). Results showed that pulse derived CO(2)-C was assimilated at a rate of 128 +/- 32 microg g shoot-C hour(-1). Dynamic samplings showed that pulse-derived (13)C concentrations in the labelled plant tissues declined by 77.4 +/- 6% after 48 hours. The rapid decline in (13)C concentrations in plant matter was the result of C loss from the plant in the form of respired CO(2) and root exudates, and dilution by subsequent unlabelled C assimilates. This novel system offers considerable potential for in situ tracer investigations.     

Structural and electronic properties of TaSi(n) (n=1-13) clusters: a relativistic density functional investigation

The TaSi(n) (n=1-13) clusters with doublet, quartet, and sextet spin configurations have been systematically investigated by a relativistic density functional theory with the generalized gradient approximation available in Amsterdam density functional program. The total bonding energies, equilibrium geometries, Mulliken populations as well as Hirshfeld charges of TaSi(n) (n=1-13) clusters are calculated and presented. The emphasis on the stabilities and electronic properties is discussed. The most stable structures of the small TaSi(n) (n=1-6) clusters and the evolutional rule of low-lying geometries of the larger TaSi(n) (n=7-13) clusters are obtained. Theoretical results indicate that the most stable structure of TaSi(n) (n=1-6) clusters keeps the similar framework as the most stable structure of Si(n+1) clusters except for TaSi(3) cluster. The Ta atom in the lowest-energy TaSi(n) (n=1-13) isomers occupies a gradual sinking site, and the site moves from convex, to flatness, and to concave with the number of Si atom varying from 1 to 13. When n=12, the Ta atom in TaSi(12) cluster completely falls into the center of the Si frame, and a cagelike TaSi(12) geometry is formed. Meanwhile, the net Mulliken and Hirsheld populations of the Ta atom in the TaSi(n) (n=1-13) clusters vary from positive to negative, manifesting that the charges in TaSi(n) (n>/=12) clusters transfer from Si atoms to Ta atom. Additionally, the contribution of Si-Si and Si-Ta interactions to the stability of TaSi(n) clusters is briefly discussed. Furthermore, the investigations on atomic averaged binding energies and fragmentation energies show that the TaSi(n) (n=2,3,5,7,10,11,12) clusters have enhanced stabilities. Compared with pure silicon clusters, a universal narrowing of highest occupied molecular orbital-lowest unoccupied molecular orbital gap in TaSi(n) clusters is found.     

Functional stability of a mixed microbial consortium producing PHA from waste carbon sources

Polyhydroxyalkanoates (PHAs) represent an environmentally effective alternative to synthetic thermoplastics; however, current production practices are not sustainable. In this study, PHA production was accomplished in sequencing batch bioreactors utilizing real wastewaters and mixed microbial consortia from municipal activated sludge as inoculum. Polymer production reached 85, 53, and 10% of the cell dry weight from methanol-enriched pulp and paper mill foul condensate, fermented municipal primary solids, and biodiesel wastewater, respectively. Using denaturing gradient gel electrophoresis of 16S-rDNA from polymerase chain reaction-amplified DNA extracts, distinctly different communities were observed between and within wastewaters following enrichment. Most importantly, functional stability was maintained despite differing and contrasting microbial populations.     

Limitations in detection of 15N incorporation by mass spectrometry in protein-based stable isotope probing (protein-SIP)

The method of protein-based stable isotope probing (protein-SIP) has previously been shown to allow the modeling of carbon fluxes in microbial communities, thus tackling one of the key questions in microbial ecology. The method allows the analysis of stable isotope distribution in peptides, revealing metabolic activities of the species present in an ecosystem. Besides carbon, an application of protein-SIP with nitrogen is of interest for resolving the nitrogen fluxes in microbial communities. Thus, the sensitivity and reliability of a protein-SIP approach employing ¹⁵N was analyzed. For this, cultivations of Pseudomonas fluorescens ATCC 17483 with different ratios of ¹⁴N/¹⁵N were performed, from 10 % down to 0.1 % ¹⁵N. After incubation leading to complete labeling of biomass, proteins were extracted and separated by one-dimensional gel electrophoresis, followed by tryptic digest and UPLC Orbitrap MS/MS analysis. ¹⁵N relative isotope abundance (RIA) was calculated based on isotopic patterns from identified peptides in mass spectra. Proteomics data have been deposited to ProteomeXchange with identifier PXD000127. The distribution of ¹⁵N RIA values among peptides was analyzed in samples with different ¹⁵N amount, and potential causes for variations within individual samples of either technical or biological origin were investigated. Using a number of 50 peptides, significant differences (p?≤?0.05) in ¹⁵N incorporation were found between samples of different ¹⁵N RIA down to 0.1 %. The study demonstrates that protein-SIP using ¹⁵N is sufficiently sensitive for quantitative investigation of microbial activity in nitrogen cycling processes.     

Isolation and characterization of native nuclear deoxyribonucleic acid from tobacco leaves]

Tobacco nuclear DNA (nDNA) was isolated from tobacco leaf nuclei which were prepared according to our previously published procedure [8]. The nDNA was characterized by base analysis, absorption spectrophotometry, analytical CsCl density gradient equilibrium centrifugation and by its melting behaviour. The results show that the isolated tobacco nDNA is native, high molecular weight DNA, which is free of detectable amounts of chloroplast DNA, RNA, protein and polysaccharides. From its melting behaviour it was concluded that tobacco nDNA can be placed close to calf thymus DNA with respect to intramolecular heterogeneity. Experiments on the partial and complete denaturation of tobacco nDNA and its ability to renature are also reported.     

Compound-specific delta13C analysis of individual amino sugars--a tool to quantify timing and amount of soil microbial residue stabilization

There is strong scientific evidence that microbial residues such as amino sugars may be stabilized in soil. However, up to now, no investigation has been carried out to quantify both the amount and timing of such stabilization. This is primarily due to methodological constraints, because it is not possible to differentiate between stabilized (old) and recently produced (new) amino sugars when these biomarkers are conventionally analyzed, e.g. by means of gas chromatography and flame ionization detection. Therefore, the aim of the present study was to test whether compound-specific isotope analysis (delta13C) of amino sugars extracted from soil could be used to differentiate between old and new microbial residues. For this aim a method for the delta13C analysis of individual amino sugars was developed and optimized. First results of delta13C values of glucosamine, galactosamine, mannosamine, and muramic acid in soil samples from two different ecological studies are presented, clearly indicating that discrimination between soil inherent and newly formed amino sugars is possible in stable isotope labeling experiments. Our results further showed that, in the short term (within 1 month), only few amino sugars were built, thus making highly 13C-enriched substrates necessary for the quantification of new amino sugar production and for the determination of amino sugar turnover rates.     

Optimisation of derivatisation procedures for the determination of delta13C values of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry

Compound-specific stable carbon isotope analysis of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is a highly selective and sensitive method for probing the biosynthetic/diagenetic pathways, pool size and turnover rates of proteins, previously intractable to bulk isotope analyses. However, amino acids are polyfunctional, non-volatile compounds which require derivatisation prior to GC analysis. While a wide range of derivatives exist for the GC analysis of amino acids only a handful have been utilised for their GC/C/IRMS analysis. Significantly, none of those derivatives currently employed appear completely satisfactory and a thorough assessment of their relative utility is lacking. Seven derivatives (three previously reported and four novel) for obtaining delta(13)C values of amino acids via GC/C/IRMS analysis were compared. More specifically, standard mixtures of 15 protein amino acids were converted into N-acetylmethyl (NACME) esters, N-acetyl n-propyl (NANP) esters, N-acetyl i-propyl (NAIP) esters, N-trifluoroacetyl-i-propyl (TFA-IP) esters, N-pivaloyl methyl (NPME) esters, N-pivaloyl n-propyl (NPNP) esters and N-pivaloyl i-propyl (NPIP) esters. Each derivative was assessed with respect to its applicability to carbon isotope determinations of all the common alpha-amino acids, reaction yield, chromatographic resolution, stability, analyte-to-derivative carbon ratio, kinetic isotope effects and errors associated with their carbon isotope determinations. The NACME derivative was concluded to be the preferred derivative mainly due to the highest analyte-to-derivative carbon ratio being achieved, resulting in the lowest analytical errors for amino acid delta(13)C value determinations, ranging from +/-0.6 per thousand for phenylalanine, leucine and isoleucine to +/-1.1 per thousand for serine and glycine.     

One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles

In the present study, we describe a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tissue organelles, we performed one-step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing-thawing overnight at -20 degrees C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.     

Isolation of neutral and acidic lipid biomarker classes for compound-specific-carbon isotope analysis by means of solvent extraction and normal-phase high-performance liquid chromatography

An analytical method for the quantitative determination of neutral and acidic lipid biomarkers in particulate and sediment samples has been developed. The method involves a first step with solvent extraction to isolate the neutral from the acidic compounds and a second step using normal-phase HPLC on a Nucleosil silica column to separate four different classes of neutral compounds: (1) aliphatic hydrocarbons, (2) aromatic hydrocarbons, (3) ketone compounds and (4) sterol and alcohol compounds. Recoveries of the individual spiked lipid biomarkers for the whole analytical procedure ranged from 88 to 106% for fatty acids, from 50 to 60% for aliphatic hydrocarbons (> or = n-C17), from 50 to 60% for polycyclic aromatic hydrocarbons (PAHs) (> or = 3 rings), 83% for friedelin and 60-80% for the sterol and alcohol compounds. The isolated compound classes were analysed by gas chromatography-combustion-isotope ratio mass spectrometry to measure the stable carbon isotope ratios in the individual compounds. The method enables the isolation of compound classes without fractionation for compound-specific carbon isotope analysis (delta13C). This analytical protocol has been applied, and proved suitable, for the determination of lipid biomarkers (sterols, fatty alcohols and fatty acids) in marine particulate material and for the determination of PAHs in sediment samples.     

A rapid method for fractionation and purification of a 92 kDa cartilage glycoprotein

Matrix glycoproteins are among the main components that contribute to the properties of cartilage. In this article we report on the development of a rapid method for the fractionation and purification of a 92 kDa glycoprotein from chick sternal cartilage. The developed procedure involves ion-exchange chromatography on DEAE-Sephacel, gel permeation chromatography on Sepharose CL-6B and semi-preparative SDS-polyacrylamide gel electrophoresis. Identification of protein was performed by western blotting using specific antibodies and purity by capillary electrophoresis. The proposed method is superior to those previously published since it eliminates the step of density gradient centrifugation.     

Density gradient electrophoresis of cells in a reversible gel

Density gradient electrophoresis permits the separation of cell types according to surface charge density with high resolution. Any source of flow compromises the resolving power of density gradient electrophoresis. Although procedures have been devised to successfully counteract electroosmotic and convective flows, the final collection of separands requires that they be pumped out of the electrophoresis column. Experiments were therefore designed to test the hypothesis that this flow could also be eliminated by trapping the separated bands in a gel, from which they could be collected by slicing the gel cylinder. Glutaraldehyde-fixed rat and rabbit erythrocytes were used as test particles in a phosphate-buffered isotonic Ficoll-sucrose density gradient in a 2.2 cm diameter, thermostated vertical glass column that could be opened at both ends. Two types of agarose were used as gel polymers: Electrophoresis grade agarose (J.T. Baker Chemical Co.) at final concentrations of 0.1 to 0.25% and SeaPrep ultralow gelling agarose (Marine Colloids Div., FMC Corp.) at a final concentration of 1.0%. Electrophoretic separability of the test particles and fluid stability were tested independently at 55 degrees C and 32 degrees C at which the two agaroses were, respectively, liquid. The experiments demonstrated that the higher temperatures required and the presence of agarose compromised neither the stability of the density gradient nor the migration properties of the cells, and cells can be separated in a sol at a temperature that is compatible with cell viability.     

Isotope ratio monitoring of small molecules and macromolecules by liquid chromatography coupled to isotope ratio mass spectrometry

In the field of isotope ratio mass spectrometry, the introduction of an interface allowing the connection of liquid chromatography (LC) and isotope ratio mass spectrometry (IRMS) has opened a range of new perspectives. The LC interface is based on a chemical oxidation, producing CO2 from organic molecules. While first results were obtained from the analysis of low molecular weight compounds, the application of compound-specific isotope analysis by irm-LC/MS to other molecules, in particular biomolecules, is presented here. The influence of the LC flow rate on the CO2 signal and on the observed delta13C values is demonstrated. The limits of quantification for angiotensin III and for leucine were 100 and 38 pmol, respectively, with a standard deviation of the delta13C values better than 0.4 per thousand. Also, accuracy and precision of delta13C values for elemental analyser-IRMS and flow injection analysis-IRMS (FIA-LC/MS) were compared. For compounds with molecular weights ranging from 131 to 66,390 Da, precision was better than 0.3 per thousand, and accuracy varied from 0.1 to 0.7 per thousand. In a second part of the work, a two-dimensional (2D)-LC method for the separation of 15 underivatised amino acids is demonstrated; the precision of delta13C values for several amino acids by irm-LC/MS was better than 0.3 per thousand at natural abundance. For labelled mixtures, the coefficient of variation was between 1% at 0.07 atom % excess (APE) for threonine and alanine, and around 10% at 0.03 APE for valine and phenylalanine. The application of irm-LC/MS to the determination of the isotopic enrichment of 13C-threonine in an extract of rat colon mucosa demonstrated a precision of 0.5 per thousand, or 0.001 atom %.