Benchmarking Non-Targeted Metabolomics Using Yeast-Derived Libraries.

Non-targeted analysis by high-resolution mass spectrometry (HRMS) is an essential discovery tool in metabolomics. To date, standardization and validation remain a challenge. Community-wide accepted cost-effective benchmark materials are lacking. In this work, we propose yeast (Pichia pastoris) extracts derived from fully controlled fermentations for this purpose. We established an open-source metabolite library of >200 identified metabolites based on compound identification by accurate mass, matching retention times, and MS/MS, as well as a comprehensive literature search. The library includes metabolites from the classes of (1) organic acids and derivatives (2) nucleosides, nucleotides, and analogs, (3) lipids and lipid-like molecules, (4) organic oxygen compounds, (5) organoheterocyclic compounds, (6) organic nitrogen compounds, and (7) benzoids at expected concentrations ranges of sub-nM to µM. As yeast is a eukaryotic organism, key regulatory elements are highly conserved between yeast and all annotated metabolites were also reported in the human metabolome database (HMDB). Orthogonal state-of-the-art reversed-phase (RP-) and hydrophilic interaction chromatography mass spectrometry (HILIC-MS) non-targeted analysis and authentic standards revealed that 104 out of the 206 confirmed metabolites were reproducibly recovered and stable over the course of three years when stored at -80 °C. Overall, 67 out of these 104 metabolites were identified with comparably stable areas over all three yeast fermentation and are the ideal starting point for benchmarking experiments. The provided yeast benchmark material enabled not only to test for the chemical space and coverage upon method implementation and developments but also allowed in-house routines for instrumental performance tests. Transferring the quality control strategy of proteomics workflows based on the number of protein identification in HeLa extracts, metabolite IDs in the yeast benchmarking material can be used as metabolomics quality control. Finally, the benchmark material opens new avenues for batch-to-batch corrections in large-scale non-targeted metabolomics studies.

Tetracycline Antibiotics Induce Host-Dependent Disease Tolerance to Infection.

Several classes of antibiotics have long been known to have beneficial effects that cannot be explained strictly on the basis of their capacity to control the infectious agent. Here, we report that tetracycline antibiotics, which target the mitoribosome, protected against sepsis without affecting the pathogen load. Mechanistically, we found that mitochondrial inhibition of protein synthesis perturbed the electron transport chain (ETC) decreasing tissue damage in the lung and increasing fatty acid oxidation and glucocorticoid sensitivity in the liver. Using a liver-specific partial and acute deletion of Crif1, a critical mitoribosomal component for protein synthesis, we found that mice were protected against sepsis, an observation that was phenocopied by the transient inhibition of complex I of the ETC by phenformin. Together, we demonstrate that mitoribosome-targeting antibiotics are beneficial beyond their antibacterial activity and that mitochondrial protein synthesis inhibition leading to ETC perturbation is a mechanism for the induction of disease tolerance.

Chemical imaging and assessment of cadmium distribution in the human body.

The scientific interest in cadmium (Cd) as a human health damaging agent has significantly increased over the past decades. However, particularly the histological distribution of Cd in human tissues is still scarcely defined. Using inductively coupled plasma-mass spectrometry (ICP-MS), we determined the concentration of Cd in 40 different human tissues of four body donors and provided spatial information by elemental imaging on the microscopic distribution of Cd in 8 selected tissues by laser ablation (LA)-ICP-MS. ICP-MS results show that Cd concentrations differ by a factor of 20 000 between different tissues. Apart from the well know deposits in kidney, bone, and liver, our study provides evidence that muscle and adipose tissue are underestimated Cd pools. For the first time, we present spatially resolved Cd distributions in a broad panel of human soft tissues. The defined histological structures are mirrored by sharp cut differences in Cd concentrations between neighboring tissue types, particularly in the rectum, testis, and kidneys. The spatial resolution of the Cd distribution at microscopic level visualized intratissue hot spots of Cd accumulation and is suggested as a powerful tool to elucidate metal based toxicity at histological level.

Electrophoretic separation techniques and their hyphenation to mass spectrometry in biological inorganic chemistry.

Electrophoretic methods have been widely applied in research on the roles of metal complexes in biological systems. In particular, CE, often hyphenated to a sensitive MS detector, has provided valuable information on the modes of action of metal-based pharmaceuticals, and more recently new methods have been added to the electrophoretic toolbox. The range of applications continues to expand as a result of enhanced CE-to-MS interfacing, with sensitivity often at picomolar level, and evolved separation modes allowing for innovative sample analysis. This article is a followup to previous reviews about CE methods in metallodrug research (Electrophoresis, 2003, 24, 2023-2037; Electrophoresis, 2007, 28, 3436-3446; Electrophoresis, 2012, 33, 622-634), also providing a comprehensive overview of metal species studied by electrophoretic methods hyphenated to MS. It highlights the latest CE developments, takes a sneak peek into gel electrophoresis, traces biomolecule labeling, and focuses on the importance of early-stage drug development.

Combination of cadmium and high cholesterol levels as a risk factor for heart fibrosis.

The deleterious effects of increased cadmium (Cd) serum levels on the cardiovascular system are proven by epidemiological and basic science studies. Cd exposure of animals and humans is known to impair myocardial function, possibly leading to heart failure. This study aims at investigating the effect of Cd treatment on the cardiac system with emphasis on the combined effect of Cd and high serum cholesterol levels as an important cardiovascular risk factor. Detailed analyses of Cd-induced effects on the heart of ApoE-/- mice fed a high fat diet (HFD), ApoE-/- mice fed a normal diet (ND), and C57BL/6J mice fed a ND revealed proinflammatory and fibrotic changes in the presence of cellular hypertrophy but in the absence of organ hypertrophy. Hypercholesterolemia in ApoE-/- mice alone and in combination with Cd treatment resulted in significant cardiomyocyte cell death. Based on further analyses of heart sections, we conclude that severe hypercholesterolemia in combination with ApoE-/- genotype as well as Cd treatment results in necrotic cardiomyocyte death. These data were supported by in vitro experiments showing a Cd-induced depolarization of the mitochondrial membrane and the permeabilization of the plasma membrane arguing for the occurrence of Cd-induced necrotic cell death. In summary, we were able to show for the first time that the combination of high cholesterol and Cd levels increase the risk for heart failure through cardiac fibrosis. This observation could in part be explained by the dramatically increased deposition of Cd in the hearts of ApoE-/- mice fed a HFD.

A systematic capillary electrophoresis study on the effect of the buffer composition on the reactivity of the anticancer drug cisplatin to the DNA model 2'-deoxyguanosine 5'-monophosphate (dGMP).

The development of DNA-targeted next-generation platinum-based anticancer chemotherapeutics is often accompanied by studies on the reactivity to DNA models. However, the incubation conditions used in literature vary widely, and some of the buffer/salts used are known to form complexes with Pt. Such coordination can influence the binding process and also the adducts formed. In a systematic approach, studies on the binding of cisplatin (1 mM) to dGMP (2 mM) in a series of different incubation solutions of relevance to biological systems are reported, employing capillary zone electrophoresis (CZE) with UV/vis and electrospray ionization-mass spectrometric (ESI-MS) detectors. Kinetic experiments performed with CZE-UV showed a high reactivity of dGMP to cisplatin in pure water (τ(1/2) = 4.1 ± 0.7 h) but a significantly slowed down in a solution containing a carbonate/phosphate buffer supplemented with NaCl, resulting in a half-life of dGMP of 25 ± 3 h. Especially carbonate had a major impact on the binding, though no coordination to the metal center was detectable with the methods used. The only adducts containing buffer components were (phosphate)Pt and tris(ammine)Pt species, as identified by means of CZE-ESI-MS, in addition to the main adduct [Pt(NH3)2(dGMP)2 - 4H(+)](2-) and other less abundant Pt-containing species.

Capillary zone electrophoresis and capillary zone electrophoresis-electrospray ionization mass spectrometry studies on the behavior of anticancer cis- and trans-[dihalidobis(2-propanone oxime)platinum(II)] complexes in aqueous solutions.

Anticancer chemotherapeutics with new modes of action are in great demand to overcome adverse effects, resistance problems and a limited application range. Among other approaches, trans-configured analogs of the established chemotherapy drug cisplatin show promising results in biological model systems. Herein we report on comparative studies on the stability of cis- and trans-[dihalidobis(2-propanone oxime)platinum(II)] (halido=Cl(-), Br(-), I(-)) complexes in phosphate buffer, using capillary zone electrophoresis (CZE) and CZE hyphenated to electrospray ionization-mass spectrometry (CZE-ESI-MS). The used conditions simulate those in the cytoplasm, being of importance for the activation of platinum anticancer agents for their reaction with DNA, the ultimate target. The configuration of the Pt center, i.e., cis or trans, accounts for the differing degradation kinetics of the compounds and in addition a significant influence of the halido leaving group was observed, with in case of the cis complexes pseudo first order rate constants of 0.268, 0.191 and 0.142h(-1) for Br(-), Cl(-), and I(-), respectively. Degradation of the trans isomers was significantly faster compared to their cis-configured counterparts also leading to different products which were characterized by hyphenation of CZE to ESI-MS.

Cellular accumulation and DNA interaction studies of cytotoxic trans-platinum anticancer compounds.

Forty years after the discovery of the anticancer effects of cisplatin, scientists are still pursuing the development of platinum complexes with improved properties regarding side effects and resistance, which are two main problems in cisplatin treatment. Among these compounds, trans-configured platinum complexes with oxime ligands emerged as a new class with features distinct from those of established anticancer agents, including different DNA binding behavior, increased cellular accumulation, and a different pattern of protein interaction. We report herein on the reactivity with biomolecules of three novel pairs of cis- and trans-configured acetone oxime platinum(II) complexes and one pair of 3-pentanone oxime platinum(II) complexes. Cellular accumulation experiments and in vitro DNA platination studies were performed and platinum contents were determined by inductively coupled plasma mass spectrometry. The trans-configured complexes were accumulated in SW480 cells in up to 100 times higher amounts than cisplatin and up to 50 times higher amounts than their cis-configured counterparts; r (b) values (number of platinum atoms per nucleotide) were more than tenfold increased in cells treated with trans complexes compared with cells treated with cisplatin. The interaction of the complexes with DNA was studied in cell-free experiments with plasmid DNA (pUC19), in capillary zone electrophoresis with the DNA model 2-deoxyguanosine 5'-monophosphate, and in in vitro experiments showing the degree of DNA damage in the comet assay. Whereas incubation with cis compounds did not induce degradation of DNA, the trans complexes led to pronounced strand cleavage.