Profiling Thiol Metabolites and Quantification of Cellular Glutathione Using FT-ICR-MS Spectrometry

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4 ± 11.5 and 10.1 ± 4.0 nmol/mg protein, respectively

Chemoselective Detection and Discrimination of Carbonyl-Containing Compounds in Metabolite Mixtures by \u3csup\u3e1\u3c/sup\u3eH-Detected \u3csup\u3e15\u3c/sup\u3eN Nuclear Magnetic Resonance

NMR spectra of mixtures of metabolites extracted from cells or tissues are extremely complex, reflecting the large number of compounds that are present over a wide range of concentrations. Although multidimensional NMR can greatly improve resolution as well as improve reliability of compound assignments, lower abundance metabolites often remain hidden. We have developed a carbonyl-selective aminooxy probe that specifically reacts with free keto and aldehyde functions, but not carboxylates. By incorporating 15N in the aminooxy functional group, 15N-edited NMR was used to select exclusively those metabolites that contain a free carbonyl function while all other metabolites are rejected. Here, we demonstrate that the chemical shifts of the aminooxy adducts of ketones and aldehydes are very different, which can be used to discriminate between aldoses and ketoses, for example. Utilizing the 2-bond or 3-bond 15N-1H couplings, the 15N-edited NMR analysis was optimized first with authentic standards and then applied to an extract of the lung adenocarcinoma cell line A549. More than 30 carbonyl-containing compounds at NMR-detectable levels, six of which we have assigned by reference to our database. As the aminooxy probe contains a permanently charged quaternary ammonium group, the adducts are also optimized for detection by mass spectrometry. Thus, this sample preparation technique provides a better link between the two structural determination tools, thereby paving the way to faster and more reliable identification of both known and unknown metabolites directly in crude biological extracts

Same data, different conclusions: Radical dispersion in empirical results when independent analysts operationalize and test the same hypothesis

In this crowdsourced initiative, independent analysts used the same dataset to test two hypotheses regarding the effects of scientists’ gender and professional status on verbosity during group meetings. Not only the analytic approach but also the operationalizations of key variables were left unconstrained and up to individual analysts. For instance, analysts could choose to operationalize status as job title, institutional ranking, citation counts, or some combination. To maximize transparency regarding the process by which analytic choices are made, the analysts used a platform we developed called DataExplained to justify both preferred and rejected analytic paths in real time. Analyses lacking sufficient detail, reproducible code, or with statistical errors were excluded, resulting in 29 analyses in the final sample. Researchers reported radically different analyses and dispersed empirical outcomes, in a number of cases obtaining significant effects in opposite directions for the same research question. A Boba multiverse analysis demonstrates that decisions about how to operationalize variables explain variability in outcomes above and beyond statistical choices (e.g., covariates). Subjective researcher decisions play a critical role in driving the reported empirical results, underscoring the need for open data, systematic robustness checks, and transparency regarding both analytic paths taken and not taken. Implications for organizations and leaders, whose decision making relies in part on scientific findings, consulting reports, and internal analyses by data scientists, are discussed

A Facile Synthesis of (\u3cem\u3etert\u3c/em\u3e-alkoxy)amines

Tertiary alcohols react with stoichiometric BF3·Et2O and N-hydroxyphthalimide to yield N-alkoxyphthalimides. Subsequent hydrazinolyses afford the title compounds

Reductive dehydroxy coupling of 2-(hydroxymethyl)indenes to prepare ethano-bridged bis(2-indenyl) \u3cem\u3eansa\u3c/em\u3e-titanocenes

New examples of ansa-titanocenes derived from 1,2-bis(2-indenyl)ethane have been prepared. The titanium-mediated reductive coupling of 2-(hydroxymethyl)indenes provided a convenient method for substrate dimerization. Alkyl substitution of the indene ring at C(3) improved the regioselectivity of the reductive coupling to provide the ethylene bis(2-indenyl)ansa-ligands in 29–62% yield

A microfabricated preconcentration device for breath analysis

a b s t r a c t Breath analysis promises to be a noninvasive method for diagnosis of lung cancer in its early stages. Certain ketones and aldehydes in exhaled breath have been identified as indicators of lung cancer. We report a preconcentration device or preconcentrator with thousands of micropillars fabricated from a silicon wafer that have been engineered to selectively trap trace gaseous ketones and aldehydes in exhaled breath. The micropillar surfaces were functionalized with a quaternary ammonium aminooxy salt, 2-(aminooxy)ethyl-N,N,N-trimethylammonium iodide (ATM), for capturing trace carbonyl compounds flowing through the preconcentrator by means of an oximation reaction. The unreacted ATM and reacted ATM adducts were eluted out of the preconcentrator with methanol and were directly analyzed by Fourier transform-ion cyclotron resonance mass spectrometry (FTICR-MS). The preconcentration results indicate that the capture percentages of acetone depend on the molar ratio of ATM/acetone. The analysis of exhaled breath demonstrates that the preconcentrator is suitable for quantitative analysis of ketones and aldehydes in exhaled breath