Tracking isotopically labeled oxidants using boronate-based redox probes.

Reactive oxygen and nitrogen species have been implicated in many biological processes and diseases, including immune responses, cardiovascular dysfunction, neurodegeneration, and cancer. These chemical species are short-lived in biological settings, and detecting them in these conditions and diseases requires the use of molecular probes that form stable, easily detectable, products. The chemical mechanisms and limitations of many of the currently used probes are not well-understood, hampering their effective applications. Boronates have emerged as a class of probes for the detection of nucleophilic two-electron oxidants. Here, we report the results of an oxygen-18-labeling MS study to identify the origin of oxygen atoms in the oxidation products of phenylboronate targeted to mitochondria. We demonstrate that boronate oxidation by hydrogen peroxide, peroxymonocarbonate, hypochlorite, or peroxynitrite involves the incorporation of oxygen atoms from these oxidants. We therefore conclude that boronates can be used as probes to track isotopically labeled oxidants. This suggests that the detection of specific products formed from these redox probes could enable precise identification of oxidants formed in biological systems. We discuss the implications of these results for understanding the mechanism of conversion of the boronate-based redox probes to oxidant-specific products.

Salinity and stable oxygen isotope relationship in the Southwestern Atlantic: constraints to paleoclimate reconstructions.

Stable isotopes have been widely used in the literature both to discuss current ocean circulation processes, as well as to reconstitute paleoceanographic parameters. The distribution of oxygen and deuterium stable isotopes in seawater (δ18Osw and δDsw) at the Western Tropical South Atlantic border was investigated to better understand the main fractionation processes of these isotopes and establish a regional salinity and δ18Osw relation to improve the paleoceanographic knowledge in the region. This study was conducted during a quasi-synoptic oceanographic cruise in which 98 discrete seawater samples were collected in the core of the main water masses for stable isotope analysis. A strong correlation between δ18Osw and δD was found, which made it possible to extrapolate the results for δ18Osw to δD. Although it was not possible to distinguish the water masses based only on their isotopic signatures, the water masses had a strong salinity and δ18Osw relation, and compared with previous studies, a seasonal pattern was observed. Paleosalinity differences of up to 0.2 psu between Summer and Winter are reported. Considering the limitations of the current techniques to seasonally separate the samples for the paleoceanographic studies, an intermediate Mixing Line for the Tropical South Atlantic (SSS = 1.942* δ18Osw + 34.56) was proposed to reduce the estimated errors associated with these seasonal variations.

Tree-ring stable isotopes reveal twentieth-century increases in water-use efficiency of Fagus sylvatica and Nothofagus spp. in Italian and Chilean mountains.

Changes in intrinsic water use efficiency (iWUE) were investigated in Fagus sylvatica and Nothofagus spp. over the last century. We combined dendrochronological methods with dual-isotope analysis to investigate whether atmospheric changes enhanced iWUE of Fagus and Nothofagus and tree growth (basal area increment, BAI) along latitudinal gradients in Italy and Chile. Post-maturation phases of the trees presented different patterns in δ13C, Δ13C, δ18O, Ci (internal CO2 concentration), iWUE, and BAI. A continuous enhancement in isotope-derived iWUE was observed throughout the twentieth century, which was common to all sites and related to changes in Ca (ambient CO2 concentration) and secondarily to increases in temperature. In contrast to other studies, we observed a general increasing trend of BAI, with the exception of F. sylvatica in Aspromonte. Both iWUE and BAI were uncoupled with the estimated drought index, which is in agreement with the absence of enduring decline in tree growth. In general, δ13C and δ18O showed a weak relationship, suggesting the major influence of photosynthetic rate on Ci and δ13C, and the minor contribution of the regulation of stomatal conductance to iWUE. The substantial warming observed during the twentieth century did not result in a clear pattern of increased drought stress along these latitudinal transects, because of the variability in temporal trends of precipitation and in specific responses of populations.

Mass spectrum patterns of 18O-tagged peptides labeled by enzyme-catalyzed oxygen exchange.

(18)O-labeling of peptides is a technique widely and routinely applied in the protein chemistry laboratories. The rate of (18)O incorporation at the carboxyl terminus of peptides via enzyme-catalyzed oxygen exchange fluctuates from peptide to peptide. This fluctuation is mostly attributed to enzyme-substrate different affinity. The final distributions of the (18)O(0)-, (18)O(1)-, and (18)O(2)-tagged peptides remain unpredictable though usually constrained to binomial proportions. It is proved here that this constraint can sometimes be a poor model. A more general model is then derived which predicts linear paths for digestion in H(2)(18)O-enriched water while confining binomial proportions to postdigestion labeling. Both subderived models are simple in structure and relevant for the current software development in the analysis of quantitative shotgun proteomics data. Accuracy and time dependency are examined and compared with actual labeled-digest data.

Detection and characterization of cholesterol-oxidized products using HPLC coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry.

Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3beta-hydroxycholest-5-ene-7alpha-hydroperoxide (7alpha-OOH), 3beta-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH), 3beta-hydroxycholest-4-ene-6alpha-hydroperoxide (6alpha-OOH), 3beta-hydroxycholest-4-ene-6beta-hydroperoxide (6beta-OOH), and 3beta-hydroxy-5beta-hydroxy-B-norcholestane-6beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of H(2)O molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers. This is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.