Relative Isotopic Abundances Research Articles

Stellar abundances and presolar grains trace the nucleosynthetic origin of molybdenum and ruthenium

This work presents a large consistent study of molybdenum (Mo) and ruthenium (Ru) abundances in the Milky Way. These two elements are important nucleosynthetic diagnostics. In our sample of 71 Galactic metal-poor field stars, we detect Ru and/or Mo in 51 of these (59 including upper limits). The sample consists of high-resolution, high signal-to-noise spectra covering both dwarfs and giants from [Fe/H]=-0.63 down to -3.16. Thus we provide information on the behaviour of Mo I and Ru I at higher and lower metallicity than is currently known. We find a wide spread in the Mo and Ru abundances, which is typical of heavy elements. This indicates that several formation processes, in addition to high entropy winds, can be responsible for the formation of Mo and Ru. The formation processes are traced by comparing Mo and Ru to elements (Sr, Zr, Pd, Ag, Ba, and Eu) with known formation processes. We find contributions from different formation channels, namely p-, slow (s-), and rapid (r-) neutron-capture processes. Molybdenum is a highly convolved element that receives contributions from several processes, whereas Ru is mainly formed by the weak r-process as is silver. We also compare our absolute elemental stellar abundances to relative isotopic abundances of presolar grains extracted from meteorites. Their isotopic abundances can be directly linked to the formation process (e.g. r-only isotopes) providing a unique comparison between observationally derived abundances and the nuclear formation process. The comparison to abundances in presolar grains shows that the r-/s-process ratios from the presolar grains match the total elemental chemical composition derived from metal-poor halo stars with [Fe/H]~ -1.5 to -1.1 dex. This indicates that both grains and stars around and above [Fe/H]=-1.5 are equally (well) mixed and therefore do not support a heterogeneous presolar nebula... Abridged.

Automated protein turnover calculations from 15N partial metabolic labeling LC/MS shotgun proteomics data.

Protein turnover is a well-controlled process in which polypeptides are constantly being degraded and subsequently replaced with newly synthesized copies. Extraction of composite spectral envelopes from complex LC/MS shotgun proteomics data can be a challenging task, due to the inherent complexity of biological samples. With partial metabolic labeling experiments this complexity increases as a result of the emergence of additional isotopic peaks. Automated spectral extraction and subsequent protein turnover calculations enable the analysis of gigabytes of data within minutes, a prerequisite for systems biology high throughput studies. Here we present a fully automated method for protein turnover calculations from shotgun proteomics data. The approach enables the analysis of complex shotgun LC/MS 15N partial metabolic labeling experiments. Spectral envelopes of 1419 peptides can be extracted within an hour. The method quantifies turnover by calculating the Relative Isotope Abundance (RIA), which is defined as the ratio between the intensity sum of all heavy (15N) to the intensity sum of all light (14N) and heavy peaks. To facilitate this process, we have developed a computer program based on our method, which is freely available to download at http://promex.pph.univie.ac.at/protover.

Mass accuracy and isotopic abundance measurements for HR-MS instrumentation: capabilities for non-targeted analyses.

The development of automated non-targeted workflows for small molecule analyses is highly desirable in many areas of research and diagnostics. Sufficient mass and chromatographic resolution is necessary for the detectability of compounds and subsequent componentization and interpretation of ions. The mass accuracy and relative isotopic abundance are critical in correct molecular formulae generation for unknown compounds. While high-resolution instrumentation provides accurate mass information, sample complexity can greatly influence data quality and the measurement of compounds of interest. Two high-resolution instruments, an Orbitrap and a Q-TOF, were evaluated for mass accuracy and relative isotopic abundance with various concentrations of a standard mixture in four complex sample matrices. The overall average ± standard deviation of the mass accuracy was 1.06 ± 0.76ppm and 1.62 ± 1.88ppm for the Orbitrap and the Q-TOF, respectively; however, individual measurements were ± 5ppm for the Orbitrap and greater than 10ppm for the Q-TOF. Relative isotopic abundance measurements for A + 1 were within 5% of the theoretical value if the intensity of the monoisotopic peak was greater than 1E7 for the Orbitrap and 1E5 for the Q-TOF, where an increase in error is observed with a decrease in intensity. Furthermore, complicating factors were found in the data that would impact automated data analysis strategies, including coeluting species that interfere with detectability and relative isotopic abundance measurements. The implications of these findings will be discussed with an emphasis on reasonable expectations from these instruments, guidelines for experimental workflows, data analysis considerations, and software design for non-targeted analyses.

Power of isotopic fine structure for unambiguous determination of metabolite elemental compositions: In silico evaluation and metabolomic application

In mass spectrometry (MS)-based metabolomics studies, reference-free identification of metabolites is still a challenging issue. Previously, we demonstrated that the elemental composition (EC) of metabolites could be unambiguously determined using isotopic fine structure, observed by ultrahigh resolution MS, which provided the relative isotopic abundance (RIA) of 13C, 15N, 18O, and 34S. Herein, we evaluated the efficacy of the RIA for determining ECs based on the MS peaks of 20,258 known metabolites. The metabolites were simulated with a ≤25% error in the isotopic peak area to investigate how the error size effect affected the rate of unambiguous determination of the ECs. The simulation indicated that, in combination with reported constraint rules, the RIA led to unambiguous determination of the ECs for more than 90% of the tested metabolites. It was noteworthy that, in positive ion mode, the process could distinguish alkali metal-adduct ions ([M+Na]+ and [M+K]+). However, a significant degradation of the EC determination performance was observed when the method was applied to real metabolomic data (mouse liver extracts analyzed by infusion ESI), because of the influence of noise and bias on the RIA. To achieve ideal performance, as indicated in the simulation, we developed an additional method to compensate for bias on the measured ion intensities. The method improved the performance of the calculation, permitting determination of ECs for 72% of the observed peaks. The proposed method is considered a useful starting point for high-throughput identification of metabolites in metabolomic research.

Bioremediation of oil-polluted soils: using the [13C]/[12C] ratio to characterize microbial products of oil hydrocarbon biodegradation

We compared data on the extent of bioremediation in soils polluted with oil. The data were obtained using conventional methods of hydrocarbon determination: extraction gas chromatography-mass spectrometry, extraction IR spectroscopy, and extraction gravimetry. Due to differences in the relative abundances of the stable carbon isotopes (13C/12C) in oil and in soil organic matter, these ratios could be used as natural isotopic labels of either substance. Extraction gravimetry in combination with characteristics of the carbon isotope composition of organic products in the soil before and after bioremediation was shown to be the most informative approach to an evaluation of soil bioremediation. At present, it is the only method enabling quantification of the total petroleum hydrocarbons in oil-polluted soil, as well as of the amounts of hydrocarbons remaining after bioremediation and those microbially transformed into organic products and biomass.

Evaluation of the radioactive Cs concentration in brown rice based on the K nutritional status of shoots

The radioactive cesium ((134,137)Cs) concentration in brown rice is correlated with that in the straw/husk. The distribution of (134,137)Cs, resembles that of potassium (K), a homologous element of Cs, in the rice plant body. The relative isotopic abundance of (40)K is 0.0117 %; thus, 1 g K contains 30.4 Bq ⁴⁰K, and the mass of 4,000 Bq (40)K is 0.0154 g, indicating that the K concentration can be calculated from (40)K concentration. We examined if the radioactive Cs concentration in brown rice can be estimated from (40)K concentrations in straw, and especially might be predicted from the (40)K:(134,137)Cs ratio in straw. We determined the concentrations of (40)K and radioactive Cs in straw and brown rice, and found a strong correlated-equation (y = 72.922 x(-0.759); r = 0.907) between the radioactive Cs concentration in brown rice and the ⁴⁰K:(134,137)Cs ratio in straw. The estimated-radioactive Cs concentration in brown rice can be as much as double, depending on the K nutritional status changing the ⁴⁰K:(134,137)Cs ratio in straw. We herein propose a nutritional diagnosis that radioactive Cs concentrations in brown rice can be predicted from the ⁴⁰K:(134,137)Cs ratio in shoots.

LA-ICP-MS for Pu source identification at Mayak PA, the Urals, Russia

Information on Pu in environmental samples is traditionally based on the determination of the (240+239)Pu activity via Alpha Spectrometry (AS). A large number of alpha spectrometry sources (planchettes) containing radiochemically separated Pu are therefore stored worldwide and are available for further analyses. These archive samples represent a resource from which valuable information on isotopic composition of alpha emitters including Pu can be obtained. The relative abundances of Pu isotopes can be used to trace specific Pu sources and characterize the relative contributions of different Pu sources in a sample. Thus, in addition to the total (239+240)Pu activity, determination of the (240)Pu/(239)Pu ratio can provide valuable information on the nature of the Pu emitting sources. The Pu isotopic ratios can be determined by mass spectrometry techniques such as Sector Field Inductively Coupled Plasma Mass Spectrometry (SF-ICPMS) or Accelerator Mass Spectrometry (AMS) that require dissolution and complete destruction of the material deposited on the planchettes. In this study Laser Ablation (LA)-quadrupole-ICP-MS has been employed for the analysis of (239)Pu/(240)Pu ratios from alpha-planchettes prepared from samples originating from the Mayak PA nuclear facility, Russia. The results are compared with data from AMS and show that the (240)Pu/(239)Pu ratios obtained by LA-ICP-MS can be utilized to distinguish weapons-grade Pu from civil reprocessing sources. Moreover, isotope ratio mapping can also be performed across the planchettes, allowing e.g. the visualization of possible inhomogeneities in the Pu-isotope distribution on their surface. Thus, this solid sample technique can be applied to extract additional information from existing archives of samples.

Measuring chemical evolution and gravitational dependence ofαusing ultraviolet Fe v and Ni v transitions in white-dwarf spectra

In this paper, we present the details of the ab initio high-precision configuration interaction and many-body perturbation theory calculations that were used by Berengut et al. [Phys. Rev. Lett. 111, 010801 (2013)] to place limits on the dependence of the fine-structure constant, $\ensuremath{\alpha}$, on the gravitational field of the white-dwarf star G191-B2B. These calculations were combined with laboratory wavelengths and spectra from the Hubble Space Telescope Imaging Spectrograph to obtain the result $\ensuremath{\Delta}\ensuremath{\alpha}/\ensuremath{\alpha}=(4.2\ifmmode\pm\else\textpm\fi{}1.6)\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}5}$ and $(\ensuremath{-}6.1\ifmmode\pm\else\textpm\fi{}5.8)\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}5}$ using Fe v and Ni v transitions, respectively. The uncertainty in these results are dominated by the uncertainty in the laboratory wavelengths. In this work we also present ab initio calculations of the isotopic shifts of the Fe v transitions. We show that improved laboratory spectra will enable determination of the relative isotope abundances in Fe v to an accuracy of $\ensuremath{\sim}$20%. Therefore this work provides a strong motivation for new laboratory measurements.

Improved determination of the Boltzmann constant using a single, fixed-length cylindrical cavity

We report improvements to our previous (Zhang et al 2011 Int. J. Thermophys. 32 1297) determination of the Boltzmann constant kB using a single 80 mm long cylindrical cavity. In this work, the shape of the gas-filled resonant cavity is closer to that of a perfect cylinder and the thermometry has been improved. We used two different grades of argon, each with measured relative isotopic abundances, and we used two different methods of supporting the resonator. The measurements with each gas and with each configuration were repeated several times for a total of 14 runs. We improved the analysis of the acoustic data by accounting for certain second-order perturbations to the frequencies from the thermo-viscous boundary layer. The weighted average of the data yielded kB = 1.380 6476 × 10−23 J K−1 with a relative standard uncertainty ur(kB) = 3.7 × 10−6. This result differs, fractionally, by (−0.9 ± 3.7) × 10−6 from the value recommended by CODATA in 2010. In this work, the largest component of the relative uncertainty resulted from inconsistent values of kB determined with the various acoustic modes; it is 2.9 × 10−6. In our previous work, this component was 7.6 × 10−6.

Structure‐oriented UHPLC‐LTQ Orbitrap‐based approach as a dereplication strategy for the identification of isoflavonoids from Amphimas pterocarpoides crude extract

Hyphenated techniques and especially ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) are nowadays widely employed in natural products research. However, the complex nature of plant extracts complicates considerably the analysis and the identification of their constituents. Nevertheless, new MS analyzers with increased resolving power and accuracy such as the orbital trap (Orbitrap) could facilitate drastically this process. The objective of this study is the development of a new structure-oriented approach based on fast UHPLC-high-resolution (HR)MS and HRMS/MS methodologies for the identification of isoflavonoids in crude extracts. In addition, aims to assist dereplication procedures, to decrease the laborious isolation steps and orient the focused isolation of compounds of interest. As a proof of concept, the methanol extract of the stem bark of Amphimas pterocarpoides (Leguminosae) was selected. Based on chromatographic (retention time, polarity) and spectrometric features (ultraviolet spectra, accurate m/z, proposed elemental composition, ring double bond equivalent, and relative isotopic abundance) as well as HRMS/MS spectra, several isoflavonoids were identified. In order to verify the proposed structures, 11 isoflavonoids were selectively isolated and unambiguously identified using 1&2D nuclear magnetic resonance techniques. Moreover, the isolated isoflavonoids were studied in HRMS/MS level, employing electrospray ionization and atmospheric pressure chemical ionization sources, in both modes. Useful information regarding their fragmentation patterns was obtained, and characteristic diagnostic ions were defined for the identification of methoxylated isoflavones, dihydroisoflavones and 5-hydroxylated isoflavonoids. Based on the current results, the proposed dereplication strategy was verified and could comprise a novel approach for the analysis of crude extracts in the future not only for isoflavonoids but also for other chemical classes of natural products.

Properties of isotope patterns and their utility for peptide identification in large‐scale proteomic experiments

In proteomic experiments, isotope patterns are routinely generated for all detected peptides. While this pattern is determined by peptide composition, it has not been evaluated as a parameter that can help in the process of peptide identification. First, we investigated how the relative isotope abundance (RIA) accuracy in proteomic data sets depends on the spectral intensity, resolution, and the number of mass spectrometry (MS) 1 scans, using an Orbitrap Velos mass spectrometer. Next, we explored the discriminatory power of isotope patterns in the context of proteome analyses of various complexities, either alone or in combination with a Mascot database search. Finally, we provide a theoretical framework for the required accuracies of both peptide mass and RIA for peptide identification. We demonstrate that the RIA error obtained in routine proteome analyses is 4-5%, and that this is only modestly influenced by spectral intensity, resolution, and the number of MS1 scans. While RIA alone has no discriminatory power, in a Mascot search isotope patterns can distinguish top scoring hits from runner-up hits in 70-95% of cases. Our theoretical approach shows that RIA accuracy needs to be ~0.2% in order to uniquely identify peptides in full proteomes. Our results demonstrate that isotope patterns can have discriminatory power when used in combination with a classical database search. Inclusion of this parameter in proteomic workflows may help to increase confidence in peptide identification, but in practical terms this will be limited to small proteomes.

Evaluation of Relative Isotopic Abundance Measurements in a Quadrupole Time-of-Flight Mass Spectrometer for Elemental Composition Determination of Natural Products in Traditional Chinese Medicine

The relative isotopic abundance (RIA) measurement errors of a quadrupole time-of-flight (Q-ToF) instrument incorporating analog-to-digital converter detectors were systemically evaluated by stochastically collecting about 200 data in positive ion mass spectrometry (MS) mode. Errors varied with peak intensities at definite spectral acquisition rates but were very close, even if peak intensities changed sharply at different spectral acquisition rates with the same concentration. Intensity thresholds were systematically defined at 1 Hz of spectral acquisition rates. RIA measurement errors were also evaluated using peak area. It seemed that peak area was better adapted for the high-intensity ions while peak intensity was suited for very low-intensity ions. Several known compounds were selected for RIA measurements for product ions in tandem mass spectropmetry (MS/MS) mode. An extract of a representative traditional Chinese medicinal, Paederia scandens was analyzed with high-performance liquid chromatography-electrospray ionization-QToF-MS/MS. The unique elemental compositions of some compounds could not be identified even with exact masses and MS/MS spectra of measured and reference compounds. RIA errors, especially of (M+2)M(-1), provided vital information for determining the elemental composition.

Lead Theft-A Study of the “Uniqueness” of Lead from Church Roofs

In the United Kingdom, theft of lead is common, particularly from churches and other public buildings with lead roofs. To assess the potential to distinguish lead from different sources, 41 samples of lead from 24 church roofs in Northamptonshire, U.K, have been analyzed for relative abundance of trace elements and isotopes of lead using X-ray fluorescence (XRF) and inductively coupled plasma mass spectrometry, respectively. XRF revealed the overall presence of 12 trace elements with the four most abundant, calcium, phosphorus, silicon, and sulfur, showing a large weight percentage standard error of the mean of all samples suggesting variation in the weight percentage of these elements between different church roofs. Multiple samples from the same roofs, but different lead sheets, showed much lower weight percentage standard errors of the mean suggesting similar trace element concentrations. Lead isotope ratios were similar for all samples. Factors likely to affect the occurrence of these trace elements are discussed.

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 (15)N was analyzed. For this, cultivations of Pseudomonas fluorescens ATCC 17483 with different ratios of (14)N/(15)N were performed, from 10 % down to 0.1 % (15)N. 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. (15)N 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 (15)N RIA values among peptides was analyzed in samples with different (15)N 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 (15)N incorporation were found between samples of different (15)N RIA down to 0.1 %. The study demonstrates that protein-SIP using (15)N is sufficiently sensitive for quantitative investigation of microbial activity in nitrogen cycling processes.

On the conversion of tritium units to mass fractions for hydrologic applications

We develop a general equation for converting laboratory-reported tritium levels, expressed either as concentrations (tritium isotope number fractions) or mass-based specific activities, to mass fractions in aqueous systems. Assuming that all tritium is in the form of monotritiated water simplifies the derivation and is shown to be reasonable for most environmental settings encountered in practice. The general equation is nonlinear. For tritium concentrations c less than 4.5×1012 tritium units (TU) – i.e. specific tritium activities<5.3×1011 Bq kg−1 – the mass fraction w of tritiated water is approximated to within 1 part per million by w ≈ c×2.22293×10−18, i.e. the conversion is linear for all practical purposes. Terrestrial abundances serve as a proxy for non-tritium isotopes in the absence of sample-specific data. Variation in the relative abundances of non-tritium isotopes in the terrestrial hydrosphere produces a minimum range for the mantissa of the conversion factor of [2.22287; 2.22300].

Analysis of iridoid glucosides from Paederia scandens using HPLC–ESI-MS/MS

Iridoid glycosides are an important class of natural products and have many biological activities. Iridoid glucosides in an extract of the plant species Paederia scandens were investigated using reversed-phase high performance liquid chromatography and electrospray quadrupole time-of-flight-type tandem mass spectrometry. The elemental composition of most of the compounds was determined by accurate mass and relative isotopic abundance (RIA) measurements. In positive ion mode, the fragmentation of [M+NH4](+) precursor ions was carried out using low energy collision-induced electrospray ionization tandem spectrometry. The neutral losses of NH3, H2O, Glc, and the side chain of the iridoid moiety were the main fragmentation patterns observed. For simple iridoid glycosides, the main differences were related to the side chains. Fragmentation of the [M-H](-)precursor ions was achieved for the compounds possibly having phenolic acid group. The connection order of the iridoid, sugar, and phenolic acid moieties, and the linkage of the 6-OH group of the sugar to the phenolic acid were unambiguously confirmed using a combination of MS/MS spectra in both positive and negative ion modes, and our previous work. For some trace dimeric iridoid glucosides, the connection order between the asperuloside and paederoside moieties was determined by the characteristic product ions; this was supported by D-labeling experiments. A total of 24 iridoid glucosides, including 14 new species, were identified or tentatively characterized based on exact mass, RIA values, tandem mass spectra, and D-labeling experiments.

Subsurface mass transport affects the radioxenon signatures that are used to identify clandestine nuclear tests

AbstractThe ratios of noble gas radioisotopes can provide critical information with which to verify that a belowground nuclear test has taken place. The relative abundance of anthropogenic isotopes is typically assumed to rely solely on their fission yield and decay rate. The xenon signature of a nuclear test is then bounded by the signal from directly produced fission xenon, and by the signal that would come from the addition of xenon from iodine precursors. Here we show that this signal range is too narrowly defined. Transport simulations were done to span the range of geological conditions within the Nevada Test Site. The simulations assume a 1 kt test and the barometric history following the nuclear test at Pahute Mesa in March 1992. Predicted xenon ratios fall outside of the typically assumed range 20% of the time and situations can arise where the ground level signal comes entirely from the decay of iodine precursors.

Stable Isotope Analysis in Primatology: A Critical Review

Stable isotope analysis has become an important tool in ecology over the last 25 years. A wealth of ecological information is stored in animal tissues in the relative abundances of the stable isotopes of several elements, particularly carbon and nitrogen, because these isotopes navigate through ecological processes in predictable ways. Stable carbon and nitrogen isotopes have been measured in most primate taxonomic groups and have yielded information about dietary content, dietary variability, and habitat use. Stable isotopes have recently proven useful for addressing more fine-grained questions about niche dynamics and anthropogenic effects on feeding ecology. Here, we discuss stable carbon and nitrogen isotope systematics and critically review the published stable carbon and nitrogen isotope data for modern primates with a focus on the problems and prospects for future stable isotope applications in primatology.

Post-run target screening strategy for ultra high performance liquid chromatography coupled to Orbitrap based veterinary drug residue analysis in animal urine

The performance of liquid chromatography coupled to high resolution mass spectrometry (LC–HRMS) post-run target screening for veterinary drug residue analysis (sulfonamides, tetracyclines and quinolones) in animal urine has been critically evaluated. It was found that retention time information still remains an essential information and that accurate masses together with relative isotopic abundance data alone are not sufficient for many residue applications. Post-run target screening requires the careful setting of parameters to achieve near zero false negative (above a defined threshold level) and a manageable numbers of false positive findings. HRMS offers many possibilities for the reduction of false positives (e.g. isotopic ratio, isotopic fine structure, exact mass of fragment ions). However, the successful use of such tools requires a sufficient ion intensity. This is often not available when trace level compounds are to be detected. Nevertheless, the proposed method is sufficiently sensitive to detect the veterinary drugs at the relevant concentration levels in urine. This means that the approach is well suited to significantly reduce the number of corresponding meat samples which have to be analyzed in a final step for the regulatory relevant quantification of residue levels in meat. The semi-quantitative screening of many samples for a large number of analytes within a short period of time requires the availability of software tools which provide fast and reliable answers.

Protein-SIP enables time-resolved analysis of the carbon flux in a sulfate-reducing, benzene-degrading microbial consortium

Benzene is a major contaminant in various environments, but the mechanisms behind its biodegradation under strictly anoxic conditions are not yet entirely clear. Here we analyzed a benzene-degrading, sulfate-reducing enrichment culture originating from a benzene-contaminated aquifer by a metagenome-based functional metaproteomic approach, using protein-based stable isotope probing (protein-SIP). The time-resolved, quantitative analysis of carbon fluxes within the community supplied with either (13)C-labeled benzene or (13)C-labeled carbonate yielded different functional groups of organisms, with their peptides showing specific time dependencies of (13)C relative isotope abundance indicating different carbon utilization. Through a detailed analysis of the mass spectrometric (MS) data, it was possible to quantify the utilization of the initial carbon source and the metabolic intermediates. The functional groups were affiliated to Clostridiales, Deltaproteobacteria and Bacteroidetes/Chlorobi. The Clostridiales-related organisms were involved in benzene degradation, putatively by fermentation, and additionally used significant amounts of carbonate as a carbon source. The other groups of organisms were found to perform diverse functions, with Deltaproteobacteria degrading fermentation products and Bacteroidetes/Chlorobi being putative scavengers feeding on dead cells. A functional classification of identified proteins supported this allocation and gave further insights into the metabolic pathways and the interactions between the community members. This example shows how protein-SIP can be applied to obtain temporal and phylogenetic information about functional interdependencies within microbial communities.