Predicting the number of sulfur atoms in peptides and small proteins based on the observed aggregated isotope distribution.

RATIONALE: Identification of peptides and proteins is a challenging task in mass spectrometry-based proteomics. Knowledge of the number of sulfur atoms can improve the identification of peptides and proteins. METHODS: In this article, we propose a method for the prediction of S-atoms based on the aggregated isotope distribution. The Mahalanobis distance is used as dissimilarity measure to compare mass- and intensity-based features from the observed and theoretical isotope distributions. RESULTS: The relative abundance of the second and the third aggregated isotopic variants (as compared to the monoisotopic one) and the mass difference between the second and third aggregated isotopic variants are the most important features to predict the number of S-atoms. CONCLUSIONS: The mass and intensity accuracies of the observed aggregated isotopic variants are insufficient to accurately predict the number of atoms. However, using a limited set of predictions for a peptide, rather than predicting a single number of S-atoms, has a reasonably high prediction accuracy.

Mercury bioaccumulation in tilefish from the northeastern Gulf of Mexico 2 years after the Deepwater Horizon oil spill: Insights from Hg, C, N and S stable isotopes.

Mercury (Hg) concentration in fish of the Gulf of the Mexico (GoM) is a major concern due to the importance of the GoM for U.S. fisheries. The Deepwater Horizon (DWH) oil spill in April 2010 in the northern GoM resulted in large amounts of oil and dispersant released to the water column, which potentially modified Hg bioaccumulation patterns in affected areas. We measured Hg species (methylmercury (MMHg) and inorganic Hg (IHg)) concentrations, and light (C, N and S) and Hg stable isotopes in muscle and liver tissues from tilefish (Lopholatilus chamaleonticeps) sampled in 2012 and 2013 along the shelf break of the northeastern GoM. Fish located close to the mouth of the Mississippi River (MR) and northwest of the DWH well-head (47 km) showed significantly lower Hg levels in muscle and liver than fish located further northeast of the DWH (>109 km), where 98% of tilefish had Hg levels in the muscle above US consumption advisory thresholds (50% for tilefish close to the DWH). Differences in light and Hg stable isotopes signatures were observed between these two areas, showing higher δ15N, and lower δ202Hg, Δ199Hg and δ34S in fish close to the DWH/MR. This suggests that suspended particles from the MR reduces Hg bioavailability at the base of the GoM food chains. This phenomenon can be locally enhanced by the DWH that resulted in increased particles in the water column as evidenced by the marine snow layer in the sediments. On the other hand, freshly deposited Hg associated with organic matter in more oligotrophic marine waters enhanced Hg bioaccumulation in local food webs. Comparing Hg isotopic composition in liver and muscle of fish indicates specific metabolic response in fish having accumulated high levels of MMHg.

Carbon, nitrogen and sulphur isotopic fractionation in captive juvenile hooded seal (Cystophora cristata): Application for diet analysis.

RATIONALE: Intrinsic biogeochemical markers, such as stable isotope ratios of carbon, nitrogen and sulphur, are increasingly used to trace the trophic ecology of marine top predators. However, insufficient knowledge of fractionation processes in tissues continues to hamper the use of these markers. METHODS: We performed a controlled feeding experiment with eight juvenile hooded seals (Cystophora cristata) that were held on a herring-based diet (Clupea harengus) for two years. Stable isotope ratios were measured via isotope ratio mass spectrometry in three of their tissues and related to values of these markers in their diet. RESULTS: Diet-tissue isotope enrichment (trophic enrichment factor, TEF) values between dietary herring and seal tissues for carbon (Δ13 C) were +0.7 ‰ for red blood cells, +1.9 ‰ for hair and +1.1 ‰ for muscle. The TEFs for nitrogen trophic (Δ15 N) were +3.3 ‰ for red blood cells, +3.6 ‰ for hair and +4.3 ‰ for muscle. For sulphur, the Δ34 S values were +1.1 ‰ for red blood cells, +1.0 ‰ for hair and +0.9 ‰ for muscle. CONCLUSIONS: These enrichment values were greater than those previously measured in adult seals. This increase may be related to the higher rate of protein synthesis and catabolism in growing animals. This study is the first report on sulphur isotope enrichment values for a marine mammal species.

Medical applications of Cu, Zn, and S isotope effects.

This review examines recent applications of stable copper, zinc and sulfur isotopes to medical cases and notably cancer. The distribution of the natural stable isotopes of a particular element among coexisting molecular species varies as a function of the bond strength, the ionic charge, and the coordination, and it also changes with kinetics. Ab initio calculations show that compounds in which a metal binds to oxygen- (sulfate, phosphate, lactate) and nitrogen-bearing moieties (histidine) favor heavy isotopes, whereas bonds with sulfur (cysteine, methionine) favor light isotopes. Oxidized cations (e.g., Cu(ii)) and low coordination numbers are expected to favor heavy isotopes relative to their reduced counterparts (Cu(i)) and high coordination numbers. Here we discuss the first observations of Cu, Zn, and S isotopic variations, three elements closely related along multiple biological pathways, with emphasis on serum samples of healthy volunteers and of cancer patients. It was found that heavy isotopes of Zn and to an even greater extent Cu are enriched in erythrocytes relative to serum, while the difference is small for sulfur. Isotopic variations related to age and sex are relatively small. The 65Cu/63Cu ratio in the serum of patients with colon, breast, and liver cancer is conspicuously low relative to healthy subjects. The characteristic time over which Cu isotopes may change with disease progression (a few weeks) is consistent with both the turnover time of the element and albumin half-life. A parallel effect on sulfur isotopes is detected in a few un-medicated patients. Copper in liver tumor tissue is isotopically heavy. In contrast, Zn in breast cancer tumors is isotopically lighter than in healthy breast tissue. 66Zn/64Zn is very similar in the serum of cancer patients and in controls. Possible reasons for Cu isotope variations may be related to the cytosolic storage of Cu lactate (Warburg effect), release of intracellular copper from cysteine clusters (metallothionein), or the hepatocellular and biosynthetic dysfunction of the liver. We suggest that Cu isotope metallomics will help evaluate the homeostasis of this element during patient treatment, notably by chelates and blockers of Cu trafficking, and understand the many biochemical pathways in which this element is essential.

[Investigations on Sulfur and Carbon Isotopic Compositions of Potential Polluted Sources in Atmospheric PM2.5 in Nanjing Region].

Potential pollution sources of atmospheric PM2.5 in Nanjing region were collected, and sulfur and carbon isotopic compositions were determined by EA-IRMS synchronously. The results showed that δ³4S and δ¹³C values ranged from 1.8‰-3.7‰ and -25.50‰- -23.57‰ in coal soot particles; 4.6‰-9.7‰ and -26.32‰- -23.57‰ in vehicle exhaust; 5.2‰-9.9‰ and -19.30‰- -30.42‰ in straw soot particles, respectively. Besides, the δ¹³C value of dust was -13.45‰. It can be observed that sulfur isotopic compositions in coal soot were lower, while the carbon isotopic composition in dust was higher. Comparing with δ³4S and δ¹³C values in domestic and foreign polluted sources, we found that sulfur and carbon isotopes in atmospheric PM2.5 in Nanjing region presented an obvious regional characteristics. Therefore, the source spectrum of sulfur and carbon isotopic compositions in Nanjing region might provide an insight into source apportionment of atmospheric PM2.5.

Role of oxbow lakes in controlling redox geochemistry of shallow groundwater under a heterogeneous fluvial sedimentary environment in an agricultural field: Coexistence of iron and sulfate reduction.

This study aimed to extend the knowledge of the vertical distribution of redox conditions of shallow groundwater in heterogeneous fluvial sediments near oxbow lakes. For this study, we revisited the study area of Kim et al. (2009) to examine the redox zoning in details. Three multi-level samplers were installed along a flow path near two oxbow lakes to obtain vertical profiles of the subsurface geology and hydrochemical and isotopic data (δ(18)O and δD of water, δ(15)N and δ(18)O of nitrate, and δ(34)S of sulfate) of groundwater. Geologic logging showed that characteristics of the heterogeneous subsurface geology are closely related to the pattern of vertical redox zoning. Hydrochemical data in conjunction with nitrogen and sulfur isotope data show that the redox status of groundwater near oxbow lakes is controlled by denitrification, iron reduction, and sulfate reduction. The oxidizing condition of groundwater occurs in the sand-dominant alluvium located in the up-gradient of oxbow lakes, whereas the reducing condition accompanying denitrification, iron reduction, and local sulfate reduction is developed in silt-rich alluvium in and the downgradient of oxbow lakes. The occurrence of sulfate reduction was newly found in this study. However, the vertical profiles of redox-sensitive parameters show that iron reduction and sulfate reduction occur concurrently near oxbow lakes, although the measured redox potentials suggest that thermodynamic conditions are controlled by the stability of Fe(2+)/Fe-oxides. Therefore, this study shows that the redox condition of groundwater in the iron-rich zone should be carefully interpreted. For this purpose, depth-specific sampling and careful examination of sulfur isotope data will be very useful for identifying the redox processes occurring in the zone with overlapping iron reduction and sulfate reduction in heterogeneous fluvial sediments.

Chemical Assignment of Structural Isomers of Sulfur-Containing Metabolites in Garlic by Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry.

BACKGROUND: The chemical assignment of metabolites is crucial to understanding the relation between food composition and biological activity. OBJECTIVE: This study was designed to detect and chemically assign sulfur-containing metabolites by using LC-Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) in Allium plants. METHODS: Ultrahigh resolution (>250,000 full width at half-maximum) and mass accuracy (<1 mDa) by FTICR-MS allowed us to distinguish ions containing sulfur isotopes ((32)S and (34)S). RESULTS: Putative 69 S-containing monoisotopic ions (S-ions) were extracted from the metabolome data of onion (Allium cepa), green onion (Allium fistulosum), and garlic (Allium sativum) on the basis of theoretical mass differences between (32)S-ions and their (34)S-substituted counterparts and on the natural abundance of (34)S. Eight S-ions were chemically assigned by using the reference data according to the guidelines of the Metabolomics Standards Initiative. Three ions detected in garlic were assigned as derived from the isomers γ-glutamyl-S-1-propenylcysteine and γ-glutamyl-S-2-propenylcysteine and as S-2-propenylmercaptoglutathione on the basis of differences in key product ions identified in reference tandem MS spectra. CONCLUSION: The ability to discriminate between such geometric isomers will be extremely useful for the chemical assignment of unknown metabolites in MS-based metabolomics.

Nanoscale analysis of pyritized microfossils reveals differential heterotrophic consumption in the ~1.9-Ga Gunflint chert.

The 1.88-Ga Gunflint biota is one of the most famous Precambrian microfossil lagerstätten and provides a key record of the biosphere at a time of changing oceanic redox structure and chemistry. Here, we report on pyritized replicas of the iconic autotrophic Gunflintia-Huroniospora microfossil assemblage from the Schreiber Locality, Canada, that help capture a view through multiple trophic levels in a Paleoproterozoic ecosystem. Nanoscale analysis of pyritic Gunflintia (sheaths) and Huroniospora (cysts) reveals differing relic carbon and nitrogen distributions caused by contrasting spectra of decay and pyritization between taxa, reflecting in part their primary organic compositions. In situ sulfur isotope measurements from individual microfossils (δ(34)S(V-CDT) +6.7‰ to +21.5‰) show that pyritization was mediated by sulfate-reducing microbes within sediment pore waters whose sulfate ion concentrations rapidly became depleted, owing to occlusion of pore space by coeval silicification. Three-dimensional nanotomography reveals additional pyritized biomaterial, including hollow, cellular epibionts and extracellular polymeric substances, showing a preference for attachment to Gunflintia over Huroniospora and interpreted as components of a saprophytic heterotrophic, decomposing community. This work also extends the record of remarkable biological preservation in pyrite back to the Paleoproterozoic and provides criteria to assess the authenticity of even older pyritized microstructures that may represent some of the earliest evidence for life on our planet.

Sulfur and oxygen isotope tracing in zero valent iron based In situ remediation system for metal contaminants.

In the present study, controlled laboratory column experiments were conducted to understand the biogeochemical changes during the microbial sulfate reduction. Sulfur and oxygen isotopes of sulfate were followed during sulfate reduction in zero valent iron incubated flow through columns at a constant temperature of 20±1°C for 90 d. Sulfur isotope signatures show considerable variation during biological sulfate reduction in our columns in comparison to abiotic columns where no changes were observed. The magnitude of the enrichment in δ(34)S values ranged from 9.4‰ to 10.3‰ compared to initial value of 2.3‰, having total fractionation δS between biotic and abiotic columns as much as 6.1‰. Sulfur isotope fractionation was directly proportional to the sulfate reduction rates in the columns. Oxygen isotopes in this experiment seem less sensitive to microbial activities and more likely to be influenced by isotopic exchange with ambient water. A linear relationship is observed between δ(34)S and δ(18)O in biotic conditions and we also highlight a good relationship between δ(34)S and sulfate reduction rate in biotic columns.

Sulphur tracer experiments in laboratory animals using 34S-labelled yeast.

We have evaluated the use of (34)S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the (34)S-labelled yeast to laboratory animals to follow the fate and distribution of (34)S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of (34)S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.

µLC coupled to ICP-SFMS with post-column isotope dilution analysis of sulfur for absolute protein quantification.

Absolute protein quantification has become an important challenge in modern bioanalytical chemistry. Among several approaches based on mass spectrometric techniques, inductively coupled plasma (ICP) as ionisation source provides element-selective and sensitive detection of heteroatoms, and thus, a potentially emerging tool in protein analysis. In this work we applied coupling of capillary liquid chromatography (µLC) and inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) to the separation and determination of standard proteins. For quantification purposes, post-column isotope dilution of sulfur was applied and optimised for this type of hyphenated technique. Provided that the protein sequence is known (number of sulfur-containing amino acids, i.e. cysteines and methionines) the protein amount can then be directly calculated from the determined sulfur content in a certain protein fraction. In order to prove the reliability of the presented method, two different certified reference materials were analysed: CRM 393 (human apolipoprotein A-I) and CRM 486 (α-fetoprotein). For CRM 393 excellent agreement (37.0 ± 1.4 µmol L(-1)) was obtained with the certificate (37.7 ± 1.8 µmol L(-1)). However, the recovery rate for α-fetoprotein in CRM 486 was found to be about 60% indicating incomplete elution of the protein during the chromatographic separation.

The role of sulfur and sulfur isotope dilution analysis in quantitative protein analysis.

The element sulfur is almost omnipresent in all natural proteomes and plays a key role in protein quantification. Incorporated in the amino acids cysteine and methionine, it has been served as target for many protein-labeling reactions in classic quantitative proteomic approaches based on electrospray or MALDI mass spectrometry. This critical review discusses the potential and limitations of sulfur isotope dilution analysis (IDA) by inductively coupled plasma-mass spectrometry (ICP-MS) for absolute protein quantification. The development of this approach was made possible due to the improved sensitivity and accuracy of sulfur isotope ratio measurement by ICP-MS in recent years. The unique feature of ICP-MS, compound-independent ionization, enables compound (species)-unspecific sulfur IDA. This has the main advantage that only one generic sulfur standard (i.e., one isotopically labeled sulfur spike) is required to quantify each peptide or protein in a sample provided that they are completely separated in chromatography or electrophoresis and that their identities are known. The principles of this approach are illustrated with selected examples from the literature. The discussion includes also related fields of P/S and metal/S ratio measurements for the determination of phosphorylation degrees of proteins and stoichiometries in metalloproteins, respectively. Emerging new areas and future trends such as protein derivatization with metal tags for improved sensitivity of protein detection in ICP-MS are discussed.

Killed but metabolically active Salmonella typhimurium: application of a new technology to an old vector.

Previous studies have shown that attenuated salmonellae utilized as vaccine vectors engender strong immune responses; however, balancing immunogenicity with reactogenicity remains problematic. Recent work in other bacteria has shown that photochemical treatment of DNA excision repair mutants ( Delta uvrAB) renders organisms "killed but metabolically active" (KBMA). Here, we extend this concept to Salmonella typhimurium. A strain of attenuated S. typhimurium previously evaluated in human volunteers was further deleted for uvrAB genes and designated CKS362. Photochemical treatment of CKS362 resulted in significant inactivation. These KBMA organisms were metabolically active as shown by radioactive methionine incorporation and lactate dehydrogenase activity. In mice inoculated intraperitoneally, KBMA CKS362 was markedly less reactogenic and stimulated a humoral immune equivalent to its live counterpart. Because the parental strain has previously been found to elicit strong immune responses to Salmonella antigens, we propose CKS362 as a prototype strain to test the immunogenicity of KBMA organisms in humans.

The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat.

Enterovirus 71 (EV71) is a major etiological agent of hand, foot and mouth disease (HFMD). Several outbreaks in East Asia were associated with neurological complications and numerous deaths. EV71 possesses four structural proteins VP1-VP4 that are necessary in the formation of the pentameric icosahedral capsid. The viral capsid contributes to virulence, and VP1 is a prime target for EV71 vaccine development. Using yeast two-hybrid analysis, we demonstrated binding affinity between VP1 and three human proteins, i.e. ornithine decarboxylase (ODC1), gene trap ankyrin repeat (GTAR), and KIAA0697 expressed in brain tissue. These interactions were authenticated by co-immunoprecipitation experiments, and by indirect immunofluorescent confocal microscopy of transfected and EV71-infected Vero cells. The significant interaction between VP1 and ODC1 may compromise the latter's activity, and interfere with polyamine biosynthesis, growth and proliferation of EV71-infected cells. The interaction between VP1 and GTAR is noteworthy, since ankyrin proteins are associated with certain neural cell adhesion molecules and with the CRASH neurological syndrome. Given that VP1 is synthesized in large amounts during productive infection, these viral-host protein interactions may provide insights into the role of VP1 in the pathogenesis of EV71 disease and its neurological complications such as acute flaccid paralysis and encephalitis.

Sulfur and oxygen isotope fractionation during benzene, toluene, ethyl benzene, and xylene degradation by sulfate-reducing bacteria.

We examined the oxygen and sulfur isotope fractionation of sulfate during anaerobic degradation of toluene by sulfate-reducing bacteria in culture experiments with Desulfobacula toluolica as a type strain and with an enrichment culture Zz5-7 obtained from a benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated aquifer. Sulfur isotope fractionation can show considerable variation upon sulfate reduction and may react extremely sensitively to changes in environmental conditions. In contrast, oxygen isotope fractionation seems to be less sensitive to environmental changes. Our results clearly indicate that oxygen isotope fractionation is dominated by isotope exchange with ambient water. To verify our experimental results and to test the applicability of oxygen and sulfur isotope investigations under realistic field conditions, we evaluated isotope data from two BTEX-contaminated aquifers presented in the recent literature. On a field scale, bacterial sulfate reduction may be superimposed by processes such as dispersion, adsorption, reoxidation, or mixing. The dual isotope approach enables the identification of such sulfur transformation processes. This identification is vital for a general qualitative evaluation of the natural attenuation potential of the contaminated aquifer.