Basem Kanawati

High molecular diversity of extraterrestrial organic matter in Murchison meteorite revealed 40 years after its fall

Numerous descriptions of organic molecules present in the Murchison meteorite have improved our understanding of the early interstellar chemistry that operated at or just before the birth of our solar system. However, all molecular analyses were so far targeted toward selected classes of compounds with a particular emphasis on biologically active components in the context of prebiotic chemistry. Here we demonstrate that a nontargeted ultrahigh-resolution molecular analysis of the solvent-accessible organic fraction of Murchison extracted under mild conditions allows one to extend its indigenous chemical diversity to tens of thousands of different molecular compositions and likely millions of diverse structures. This molecular complexity, which provides hints on heteroatoms chronological assembly, suggests that the extraterrestrial chemodiversity is high compared to terrestrial relevant biological- and biogeochemical-driven chemical space.

Liquid chromatography-mass spectrometry in metabolomics research: mass analyzers in ultra high pressure liquid chromatography coupling.

The present review gives an introduction into the concept of metabolomics and provides an overview of the analytical tools applied in non-targeted metabolomics with a focus on liquid chromatography (LC). LC is a powerful analytical tool in the study of complex sample matrices. A further development and configuration employing Ultra-High Pressure Liquid Chromatography (UHPLC) is optimized to provide the largest known liquid chromatographic resolution and peak capacity. Reasonably UHPLC plays an important role in separation and consequent metabolite identification of complex molecular mixtures such as bio-fluids. The most sensitive detectors for these purposes are mass spectrometers. Almost any mass analyzer can be optimized to identify and quantify small pre-defined sets of targets; however, the number of analytes in metabolomics is far greater. Optimized protocols for quantification of large sets of targets may be rendered inapplicable. Results on small target set analyses on different sample matrices are easily comparable with each other. In non-targeted metabolomics there is almost no analytical method which is applicable to all different matrices due to limitations pertaining to mass analyzers and chromatographic tools. The specifications of the most important interfaces and mass analyzers are discussed. We additionally provide an exemplary application in order to demonstrate the level of complexity which remains intractable up to date. The potential of coupling a high field Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (ICR-FT/MS), the mass analyzer with the largest known mass resolving power, to UHPLC is given with an example of one human pre-treated plasma sample. This experimental example illustrates one way of overcoming the necessity of faster scanning rates in the coupling with UHPLC. The experiment enabled the extraction of thousands of features (analytical signals). A small subset of this compositional space could be mapped into a mass difference network whose topology shows specificity toward putative metabolite classes and retention time.

The Arabidopsis Glucosyltransferase UGT76B1 Conjugates Isoleucic Acid and Modulates Plant Defense and Senescence

The orphan small-molecule glycosyltransferase UGT76B1 is a novel player in plant pathogen defense that represses the salicylic acid pathway, yet enhances the jasmonic acid pathway. A nontargeted metabolome approach led to the identification of its substrate, 2-hydroxy-3-methyl-pentanoic acid, which itself was able to induce resistance against Pseudomonas syringae infection. Plants coordinate and tightly regulate pathogen defense by the mostly antagonistic salicylate (SA)- and jasmonate (JA)-mediated signaling pathways. Here, we show that the previously uncharacterized glucosyltransferase UGT76B1 is a novel player in this SA-JA signaling crosstalk. UGT76B1 was selected as the top stress-induced isoform among all 122 members of the Arabidopsis thaliana UGT family. Loss of UGT76B1 function leads to enhanced resistance to the biotrophic pathogen Pseudomonas syringae and accelerated senescence but increased susceptibility toward necrotrophic Alternaria brassicicola. This is accompanied by constitutively elevated SA levels and SA-related marker gene expression, whereas JA-dependent markers are repressed. Conversely, UGT76B1 overexpression has the opposite effect. Thus, UGT76B1 attenuates SA-dependent plant defense in the absence of infection, promotes the JA response, and delays senescence. The ugt76b1 phenotypes were SA dependent, whereas UGT76B1 overexpression indicated that this gene possibly also has a direct effect on the JA pathway. Nontargeted metabolomic analysis of UGT76B1 knockout and overexpression lines using ultra-high-resolution mass spectrometry and activity assays with the recombinant enzyme led to the ab initio identification of isoleucic acid (2-hydroxy-3-methyl-pentanoic acid) as a substrate of UGT76B1. Exogenously applied isoleucic acid increased resistance against P. syringae infection. These findings indicate a novel link between amino acid–related molecules and plant defense that is mediated by small-molecule glucosylation.

The gas-phase ozonolysis of beta-caryophyllene (C(15)H(24)). Part I: an experimental study.

The gas phase reaction of ozone with beta-caryophyllene was investigated in a static glass reactor at 750 Torr and 296 K under various experimental conditions. The reactants and gas phase products were monitored by FTIR-spectroscopy and proton-transfer-reaction mass spectrometry (PTR-MS). Aerosol formation was monitored with a scanning mobility particle sizer (SMPS) and particulate products analysed by liquid chromatography/mass spectrometry (HPLC-MS). The different reactivity of the two double bonds in beta-caryophyllene was probed by experiments with different ratios of reactants. An average rate coefficient at 295 K for the first-generation products was determined as 1.1 x 10(-16) cm(3) molecule(-1) s(-1). Using cyclohexane as scavenger, an OH-radical yield of (10.4 +/- 2.3)% was determined for the ozonolysis of the more reactive internal double bond, whereas the average OH-radical yield for the ozonolysis of the first-generation products was found to be (16.4 +/- 3.6)%. Measured gas phase products are CO, CO(2) and HCHO with average yields of (2.0 +/- 1.8)%, (3.8 +/- 2.8)% and (7.7 +/- 4.0)%, respectively for the more reactive internal double bond and (5.5 +/- 4.8)%, (8.2 +/- 2.8)% and (60 +/- 6)%, respectively from ozonolysis of the less reactive double bond of the first-generation products. The residual FTIR spectra indicate the formation of an internal secondary ozonide of beta-caryophyllene. From experiments using HCOOH as a Criegee intermediate (CI) scavenger, it was concluded that at least 60% of the formed CI are collisionally stabilized. The aerosol yield in the ozonolysis of beta-caryophyllene was estimated from the measured particle size distributions. In the absence of a CI scavenger the yield ranged between 5 and 24%, depending on the aerosol mass. The yield increases with addition of water vapour or with higher concentrations of formic acid. In the presence of HCHO, lower aerosol yields were observed. This suggests that HCOOH adds to a Criegee intermediate to form a low-volatility compound responsible for aerosol formation. The underlying reaction mechanisms are discussed and compared with the results from the accompanying theoretical paper.

UV‐B mediated metabolic rearrangements in poplar revealed by non‐targeted metabolomics

Plants have to cope with various abiotic stresses including UV-B radiation (280-315 nm). UV-B radiation is perceived by a photoreceptor, triggers morphological responses and primes plant defence mechanisms such as antioxidant levels, photoreapir or accumulation of UV-B screening pigments. As poplar is an important model system for trees, we elucidated the influence of UV-B on overall metabolite patterns in poplar leaves grown under high UV-B radiation. Combining non-targeted metabolomics with gas exchange analysis and confocal microscopy, we aimed understanding how UV-B radiation triggers metabolome-wide changes, affects isoprene emission, photosynthetic performance, epidermal light attenuation and finally how isoprene-free poplars adjust their metabolome under UV-B radiation. Exposure to UV-B radiation caused a comprehensive rearrangement of the leaf metabolome. Several hundreds of metabolites were up- and down-regulated over various pathways. Our analysis, revealed the up-regulation of flavonoids, anthocyanins and polyphenols and the down-regulation of phenolic precursors in the first 36 h of UV-B treatment. We also observed a down-regulation of steroids after 12 h. The accumulation of phenolic compounds leads to a reduced light transmission in UV-B-exposed plants. However, the accumulation of phenolic compounds was reduced in non-isoprene-emitting plants suggesting a metabolic- or signalling-based interaction between isoprenoid and phenolic pathways.

The gas-phase ozonolysis of beta-caryophyllene (C(15)H(24)). Part II: A theoretical study.

The O(3)-initiated oxidation of beta-caryophyllene, a sesquiterpene emitted in forested areas, was theoretically characterized for the first time using DFT quantum chemical calculations combined with statistical kinetic RRKM/master equation analysis and variational transition state theory. O(3)-Addition occurs primarily, >95%, on the endocyclic double bond without a barrier, leading to a total rate coefficient of 8.3 x 10(-24) T(3.05) exp(1028 K/T) cm(3) molecule(-1) s(-1), with a slight negative T-dependence. Thermal and chemically activated unimolecular reactions following this addition, including the so-called ester and hydroperoxide channels, and internal formation of the secondary ozonide, where characterized and quantified; a newly discovered reaction pathway through intersystem crossing from a dioxirane to a triplet bis(oxy) biradical intermediate is incorporated in the mechanism. The first generation product distribution at 298 K is predicted as 74% stabilized Criegee intermediates (CI), 8% OH radicals together with vinoxy-type 2-oxo alkyl radical co-products, 8% acids, 0.3% esters, and 9% CO(2) with two alkyl radical co-products. The thermalized CI can convert to the secondary ozonide in many reaction conditions, in particular the atmosphere; secondary ozonides are thus expected as dominant products of the beta-caryophyllene ozonolysis. These results are consistent with the experimental data presented in the accompanying paper (Part I). The temperature dependence and uncertainties of the product distribution are discussed. The high molecular weight oxygenated products, including beta-caryophyllonic acid and secondary ozonides, are expected to contribute to secondary organic aerosol formation.

Exploring the Arabidopsis sulfur metabolome

Sulfur plays a crucial role in protein structure and function, redox status and plant biotic stress responses. However, our understanding of sulfur metabolism is limited to identified pathways. In this study, we used a high-resolution Fourier transform mass spectrometric approach in combination with stable isotope labeling to describe the sulfur metabolome of Arabidopsis thaliana. Databases contain roughly 300 sulfur compounds assigned to Arabidopsis. In comparative analyses, we showed that the overlap of the expected sulfur metabolome and the mass spectrometric data was surprisingly low, and we were able to assign only 37 of the 300 predicted compounds. By contrast, we identified approximately 140 sulfur metabolites that have not been assigned to the databases to date. We used our method to characterize the γ-glutamyl transferase mutant ggt4-1, which is involved in the vacuolar breakdown of glutathione conjugates in detoxification reactions. Although xenobiotic substrates are well known, only a few endogenous substrates have been described. Among the specifically altered sulfur-containing masses in the ggt4-1 mutant, we characterized one endogenous glutathione conjugate and a number of further candidates for endogenous substrates. The small percentage of predicted compounds and the high proportion of unassigned sulfur compounds identified in this study emphasize the need to re-evaluate our understanding of the sulfur metabolome.

Attachment of Chloride Anion to Sugars: Mechanistic Investigation and Discovery of a New Dopant for Efficient Sugar Ionization/Detection in Mass Spectrometers

A new method for efficient ionization of sugars in the negative-ion mode of electrospray mass spectrometry is presented. Instead of using strongly hydrophobic dopants such as dichloromethane or chloroform, efficient ionization of sugars has been achieved by using aqueous HCl solution for the first time. This methodology makes it possible to use hydrophilic dopants, which are more appropriate for chromatographic separation techniques with efficient sugar ionization and detection in mass spectrometry. The interaction between chloride anions and monosaccharides (glucose and galactose) was studied by DFT in the gas phase and by implementing the polarizable continuum model (PCM) for calculations in solution at the high B3LYP/6-31+G(d,p)//B3LYP/6-311+G(2d,p) level of theory. In all optimized geometries of identified [M+Cl](-) anions, a non-covalent interaction exists. Differences were revealed between monodentate and bidentate complex anions, with the latter having noticeably higher binding energies. The calculated affinity of glucose and galactose toward the chloride anion in the gas phase and their chloride anion binding energies in solution are in excellent agreement with glucose and galactose [M+Cl](-) experimental intensity profiles that are represented as a function of the chloride ion concentration. Density functional calculations of gas-phase affinities toward chloride anion were also performed for the studied disaccharides sucrose and gentiobiose. All calculations are in excellent agreement with the experimental data. An example is introduced wherein HCl was used to effectively ionize sugars and form chlorinated adduct anions to detect sugars and glycosylated metabolites (anthocyanins) in real biological systems (Vitis vinifera grape extracts and wines), whereas they would not have been easily detectable under standard infusion electrospray mass spectrometry conditions as deprotonated species.

Ultrahigh resolution mass spectrometry-based metabolic characterization reveals cerebellum as a disturbed region in two animal models.

In the previous reports about cognitive dysfunction, cerebellum was thought to be a less affected tissue by genetic or environmental alterations in comparison to other tissues in the brain including hippocampus under the same conditions. In this work, we investigated two types of metabolomic alterations inside the cerebellum tissue. The first one addressed the differences in the metabolomics profiles between Transgenic (Tg) CRND8 of Alzheimers disease mice and non-transgenic (non-Tg) littermates. The second one addressed the metabolic differences between wild type mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and wild type mice which are not exposed to this toxic compound. For these two investigations, ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) was implemented. As a result, the significant changes of each comparison were tentatively annotated by the high mass accuracy generated from the measurements in the negative ion mode. The biosynthesis of amino acids was also enhanced pronouncedly, and perturbation of purine metabolism was also observed in Tg mice compared to non-Tg littermates. In another animal model, the reduced levels of amino acids were found whereas the intermediate levels in purine metabolism and fatty acids including fatty acid conjugated metabolites were elevated in cerebellar tissues of mice exposed to TCDD compared to control group. Collectively, it was demonstrated that FT-ICR/MS was a powerful tool for interpretation of the elemental compositions of the peaks, revealing that the metabolic perturbations in cerebellar tissues of mice were induced by either genetic manipulation or environmental factor. Therefore, the non-targeted approach, alternatively, provides various metabolic phenotypes for the systems-level mirror of the complex etiology of neurotoxicity in the cerebellum.

Automated microextraction sample preparation coupled on-line to FT-ICR-MS: application to desalting and concentration of river and marine dissolved organic matter.

AbstractSample preparation procedures are in most cases sample- and time-consuming and commonly require the use of a large amount of solvents. Automation in this regard can optimize the minimal-needed injection volume and the solvent consumption will be efficiently reduced. A new fully automated sample desalting and pre-concentration technique employing microextraction by packed sorbents (MEPS) cartridges is implemented and coupled to an ion cyclotron resonance Fourier-transform mass spectrometer (ICR-FT/MS). The performance of non-target mass spectrometric analysis is compared for the automated versus off-line sample preparation for several samples of aqueous natural organic matter. This approach can be generalized for any metabolite profiling or metabolome analysis of biological materials but was optimized herein using a well characterized but highly complex organic mixture: a surface water and its well-characterized natural organic matter and a marine sample having a highly salt charge and enabling to validate the presented automatic system for salty samples. The analysis of Suwannee River water showed selective C18-MEPS enrichment of chemical signatures with average H/C and O/C elemental ratios and loss of both highly polar and highly aromatic structures from the original sample. Automated on-line application to marine samples showed desalting and different chemical signatures from surface to bottom water. Relative comparison of structural footprints with the C18-concentration/desalting procedure however enabled to demonstrate that the surface water film was more concentrated in surface-active components of natural (fatty acids) and anthropogenic origin (sulfur-containing surfactants). Overall, the relative standard deviation distribution in terms of peak intensity was improved by automating the proposed on-line method. FigureThe efficient use of MEPS technique in automated form with high resolution FT-ICR-MS gives a selective spectrum, which reveals rich see water organic content. The shown high sensitivity of detected organic matter is due to effective desalting achieved by automated MEPS coupling to FT-ICR-MS technique.