Concept: Mass spectrometry
The contamination of Japan after the Fukushima accident has been investigated mainly for volatile fission products, but only sparsely for actinides such as plutonium. Only small releases of actinides were estimated in Fukushima. Plutonium is still omnipresent in the environment from previous atmospheric nuclear weapons tests. We investigated soil and plants sampled at different hot spots in Japan, searching for reactor-borne plutonium using its isotopic ratio (240)Pu/(239)Pu. By using accelerator mass spectrometry, we clearly demonstrated the release of Pu from the Fukushima Daiichi power plant: While most samples contained only the radionuclide signature of fallout plutonium, there is at least one vegetation sample whose isotope ratio (0.381 ± 0.046) evidences that the Pu originates from a nuclear reactor ((239+240)Pu activity concentration 0.49 Bq/kg). Plutonium content and isotope ratios differ considerably even for very close sampling locations, e.g. the soil and the plants growing on it. This strong localization indicates a particulate Pu release, which is of high radiological risk if incorporated.
Evidence of prehistoric dentistry has been limited to a few cases, the most ancient dating back to the Neolithic. Here we report a 6500-year-old human mandible from Slovenia whose left canine crown bears the traces of a filling with beeswax. The use of different analytical techniques, including synchrotron radiation computed micro-tomography (micro-CT), Accelerator Mass Spectrometry (AMS) radiocarbon dating, Infrared (IR) Spectroscopy and Scanning Electron Microscopy (SEM), has shown that the exposed area of dentine resulting from occlusal wear and the upper part of a vertical crack affecting enamel and dentin tissues were filled with beeswax shortly before or after the individual’s death. If the filling was done when the person was still alive, the intervention was likely aimed to relieve tooth sensitivity derived from either exposed dentine and/or the pain resulting from chewing on a cracked tooth: this would provide the earliest known direct evidence of therapeutic-palliative dental filling.
The metabolic phenotype varies widely due to external factors such as diet and gut microbiome composition, among others. Despite these temporal fluctuations, urine metabolite profiling studies have suggested that there are highly individual phenotypes that persist over extended periods of time. This hypothesis was tested by analyzing the exhaled breath of a group of subjects during nine days by mass spectrometry. Consistent with previous metabolomic studies based on urine, we conclude that individual signatures of breath composition exist. The confirmation of the existence of stable and specific breathprints may contribute to strengthen the inclusion of breath as a biofluid of choice in metabolomic studies. In addition, the fact that the method is rapid and totally non-invasive, yet individualized profiles can be tracked, makes it an appealing approach.
- Proceedings of the National Academy of Sciences of the United States of America
- Published about 1 year ago
More than 2 million people in the United States have myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We performed targeted, broad-spectrum metabolomics to gain insights into the biology of CFS. We studied a total of 84 subjects using these methods. Forty-five subjects (n = 22 men and 23 women) met diagnostic criteria for ME/CFS by Institute of Medicine, Canadian, and Fukuda criteria. Thirty-nine subjects (n = 18 men and 21 women) were age- and sex-matched normal controls. Males with CFS were 53 (±2.8) y old (mean ± SEM; range, 21-67 y). Females were 52 (±2.5) y old (range, 20-67 y). The Karnofsky performance scores were 62 (±3.2) for males and 54 (±3.3) for females. We targeted 612 metabolites in plasma from 63 biochemical pathways by hydrophilic interaction liquid chromatography, electrospray ionization, and tandem mass spectrometry in a single-injection method. Patients with CFS showed abnormalities in 20 metabolic pathways. Eighty percent of the diagnostic metabolites were decreased, consistent with a hypometabolic syndrome. Pathway abnormalities included sphingolipid, phospholipid, purine, cholesterol, microbiome, pyrroline-5-carboxylate, riboflavin, branch chain amino acid, peroxisomal, and mitochondrial metabolism. Area under the receiver operator characteristic curve analysis showed diagnostic accuracies of 94% [95% confidence interval (CI), 84-100%] in males using eight metabolites and 96% (95% CI, 86-100%) in females using 13 metabolites. Our data show that despite the heterogeneity of factors leading to CFS, the cellular metabolic response in patients was homogeneous, statistically robust, and chemically similar to the evolutionarily conserved persistence response to environmental stress known as dauer.
- Proceedings of the National Academy of Sciences of the United States of America
- Published almost 2 years ago
Tissue-thin parchment made it possible to produce the first pocket Bibles: Thousands were made in the 13th century. The source of this parchment, often called “uterine vellum,” has been a long-standing controversy in codicology. Use of the Latin term abortivum in many sources has led some scholars to suggest that the skin of fetal calves or sheep was used. Others have argued that it would not be possible to sustain herds if so many pocket Bibles were produced from fetal skins, arguing instead for unexpected alternatives, such as rabbit. Here, we report a simple and objective technique using standard conservation treatments to identify the animal origin of parchment. The noninvasive method is a variant on zooarchaeology by mass spectrometry (ZooMS) peptide mass fingerprinting but extracts protein from the parchment surface by using an electrostatic charge generated by gentle rubbing of a PVC eraser on the membrane surface. Using this method, we analyzed 72 pocket Bibles originating in France, England, and Italy and 293 additional parchment samples that bracket this period. We found no evidence for the use of unexpected animals; however, we did identify the use of more than one mammal species in a single manuscript, consistent with the local availability of hides. These results suggest that ultrafine vellum does not necessarily derive from the use of abortive or newborn animals with ultrathin hides, but could equally well reflect a production process that allowed the skins of maturing animals of several species to be rendered into vellum of equal quality and fineness.
BACKGROUND: Despite considerable global investigation over several decades, the roles of vitamin D in health and disease development remains convoluted. One recognised issue is the difficulty of accurately measuring the active forms of vitamin D. Advances made include some new methods addressing the potential interference by excluding epimers and isobars. However, there is no evidence that epimers are without function. Therefore, the aim of this study was to develop and validate, for the first time, a new assay to simultaneously measure levels of 6 forms of vitamin D along with two epimers. The assay was applied to multilevel certified reference material calibrators and 25 pooled human sera samples obtained from the Vitamin D External Quality Assessment Scheme (DEQAS) to demonstrate its efficiency. RESULTS: The assay is capable of simultaneously measuring 8 vitamin D analogues over the calibration ranges and LODs (in nmol/L) of: 1alpha25(OH)2D2 [0.015-1; 0.01], 1alpha25(OH)2D3 [0.1-100; 0.01], 25OHD3 [0.5-100, 0.025], 3-epi-25OHD3 [0.1-100, 0.05], 25OHD2 [0.5-100, 0.025], 3-epi-25OHD2 [0.1-100, 0.05], vitamin D3 [0.5-100, 0.05] and vitamin D2 [0.5-100, 0.05], using stanozolol-d3 as internal standard. Certified reference material calibrators and external quality control samples (DEQAS) were analysed to meet the standards outlined by National Institute of Standards and Technology (NIST). Validation steps included recovery and both precision and accuracy under inter- and intra-day variation limit of detection, and analysis of each analyte over a linear range. All validation parameters were in line with acceptable Food and Drug Administration (FDA) guidelines and the standards of the National Institute of Standards and Technology (NIST). All eight analogues were quantified with the 25OHD levels being commensurate with DEQAS data. CONCLUSIONS: This report details the application of a new LC-MS/MS based assay for the efficient analysis of eight analogues of vitamin D over a range of samples, which is a significant advance over the existing methods. Simultaneous measure of 8 vitamin D analogues does not compromise the analytical capability of the assay to quantify the commonly used biomarker (25OHD) for vitamin D status. The results demonstrate the feasibility of applying the assay in research and clinical practice that i) excludes misleading measures owing to epimers and isobars and ii) is able to quantify the excluded component to facilitate further in vivo investigation into the roles of ubiquitous epimers.
BACKGROUND: For shotgun mass spectrometry based proteomics the most computationally expensive step is in matching the spectra against an increasingly large database of sequences and their post-translational modifications with known masses. Each mass spectrometer can generate data at an astonishingly high rate, and the scope of what is searched for is continually increasing. Therefore solutions for improving our ability to perform these searches are needed. RESULTS: We present a sequence database search engine that is specifically designed to run efficiently on the Hadoop MapReduce distributed computing framework. The search engine implements the K-score algorithm, generating comparable output for the same input files as the original implementation. The scalability of the system is shown, and the architecture required for the development of such distributed processing is discussed. CONCLUSION: The software is scalable in its ability to handle a large peptide database, numerous modifications and large numbers of spectra. Performance scales with the number of processors in the cluster, allowing throughput to expand with the available resources.
The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules.
The alternate sigma factor sigH of Mycobacterium tuberculosis is expressed under stress and acts as a major regulator of several genes, including some other sigma factors and redox systems. While it is auto-regulated by its own promoter at the transcriptional level, its regulation at the post-translational level is through its cognate protein, an anti-sigma factor, RshA. Hither before RshA was believed to be a zinc-associated anti-sigma factor (ZAS) and the binding of RshA to SigH is redox dependent. Here, we show that RshA coordinates a [2Fe-2S] cluster using cysteines as ligands and native RshA has more affinity to [2Fe-2S] cluster than to zinc. Furthermore, we used amide hydrogen deuterium exchange mass spectrometry (HDX-MS), followed by site-directed mutagenesis in SigH and RshA, to elucidate the interaction mechanism of RshA and SigH and the potential role of metal ion clustering in SigH regulation. Three regions in SigH, comprising of residues 1-25, 58-69, 90-111, 115-132 and 157-196 and residues 35-57 of RshA show decreased deuterium exchange and reflect decreased solvent accessibility upon complexation with SigH. Of the three RshA mutants, created based on the HDX results, the RsHA E37A mutant shows stronger interaction with SigH, relative to WT RshA, while the H49A mutant abolishes interactions and the C(53)XXC(56)AXXA mutant has no effect on complexation with SigH. The D22A, D160A and E162 SigH mutants show significantly decreased binding to RshA and the E168A mutant completely abolished interactions with RshA, indicating that the SigH-RshA interaction is mediated by salt bridges. In addition, SigH-RshA interaction does not require clustering of metal ions. Based on our results, we propose a molecular model of the SigH-RshA interaction.
Measurement of insulin-like growth factor-1 (IGF-I) has utility for the diagnosis and management of growth disorders, but inter-assay comparison of results has been complicated by a multitude of reference standards, antibodies, detection methods, and pre-analytical preparation strategies. We developed a quantitative LC-MS method for intact IGF-I, which has advantages in throughput and complexity when compared to mass spectrometric approaches that rely on stable isotope dilution analysis of tryptic peptides. Since the method makes use of full-scan data, the assay was easily extended to provide quantitative measurement of IGF-II using the same assay protocol. The validated LC-MS assay for IGF-I and IGF-II provides accurate results across the pediatric and adult reference range and is suitable for clinical use.