Stable isotopic labeling in proteomics

Labeling of proteins and peptides with stable heavy isotopes (deuterium, carbon-13, nitrogen-15, and oxygen-18) is widely used in quantitative proteomics. These are either incorporated metabolically in cells and small organisms, or postmetabolically in proteins and peptides by chemical or enzymatic reactions. Only upon measurement with mass spectrometers holding sufficient resolution, light, and heavy labeled peptide ions or reporter peptide fragment ions segregate and their intensity values are subsequently used for quantification. Targeted use of these labels or mass tags further leads to specific monitoring of diverse aspects of dynamic proteomes. In this review article, commonly used isotope labeling strategies are described, both for quantitative differential protein profiling and for targeted analysis of protein modifications

Do fluid inclusions preserve δ18O values of hydrothermal fluids in epithermal systems over geological time? : evidence from paleo- and modern geothermal systems, Milos island, Aegean Sea

Stable isotope compositions of quartz (δ18Oquartz) and fluid inclusion waters (δ18OFI and δDFI) were analysed from Profitis Ilias, a low-sulphidation epithermal gold mineralisation deposit on Milos island Greece, to establish if δ18OFI preserve a record of paleo-geothermal processes. Previous studies show that mineralisation at Profitis Ilias resulted from extreme boiling and vaporisation and a zone located at approximately 430 m asl represents the transition between a liquid- and vapour-dominated system [Mineral. Dep. 36 (2001) 43]. The deposit is also closely associated with an active geothermal system, whose waters have a well-characterised stable isotope geochemistry [Pagel and Leroy (1991) Source, transport and deposition of metals. Balkema, Rotterdam, 107–112]. The samples were collected over an elevation interval of 440 m (210 to 650 m asl) to give information on the liquid- and vapour-segments of the paleo-system. The data show systematic variations with sample elevation. Samples from the highest elevations (c. 650 m asl) have the lightest δ18OFI (–7.3 ‰) and δDFI (–68.0 ‰) whilst the deepest (c. 210 m asl) are isotopically heavier (δ18OFI –3.7 ‰; δDFI –19.0 ‰). Relative changes in δ18OFI closely parallel those in δDFI. δ18Oquartz shows an opposite trend, from the lightest values (+13.9 ‰) at the lowest elevations to the heaviest (+15.1 ‰) at the highest. δ18OFI show correlations with other parameters. For example, variable fluid inclusion homogenisation temperatures in the vapour-dominated part of the system, correlate with a rapid shift in δDFI (–33.3 to –50.5 ‰) and δ18OFI (–4.1 to –6.2 ‰) and gold contents also increase in the same zone (up to 50 ppm). Comparable correlations in δ18Oquartz or δ18Ocalculated (estimated geothermal fluid from fluid inclusion homogenisation data) are absent. δ18Ocalculated are always 5 to 10 ‰ heavier than δ18OFI. Comparison with the modern geothermal system shows that δDFI–δ18OFI are similar. Isotope data for the modern system and fluid inclusion waters fall on linear trends sub-paralleling the meteoric water line and project towards seawater values. Numerical modelling favours kinetically controlled fractionation to explain differences in δ18Ocalculated and δ18Ofluid rather than diffusive post-trapping equilibration. The evidence suggests, that in low-temperature epithermal systems, δ18OFI may represent a better record of fluid process and the isotopic composition of the geothermal fluid than temperature-corrected quartz data

Comprehensive inter-laboratory calibration of reference materials for δ18O versus VSMOW using various on-line high-temperature conversion techniques

Internationally distributed organic and inorganic oxygen isotopic reference materials have been calibrated by six laboratories carrying out more than 5300 measurements using a variety of high-temperature conversion techniques (HTC) in an evaluation sponsored by the International Union of Pure and Applied Chemistry (IUPAC). To aid in the calibration of these reference materials, which span more than 125‰, an artificially enriched reference water (δ18O of +78.91‰) and two barium sulfates (one depleted and one enriched in 18O) were prepared and calibrated relative to VSMOW2 and SLAP reference waters. These materials were used to calibrate the other isotopic reference materials in this study. The seemingly large estimated combined uncertainties arise from differences in instrumentation and methodology and difficulty in accounting for all measurement bias. They are composed of the 3-fold standard errors directly calculated from the measurements and provision for systematic errors discussed in this paper. A primary conclusion of this study is that nitrate samples analyzed for δ18O should be analyzed with internationally distributed isotopic nitrates, and likewise for sulfates and organics. Authors reporting relative differences of oxygen-isotope ratios (δ18O) of nitrates, sulfates, or organic material should explicitly state in their reports the δ18O values of two or more internationally distributed nitrates (USGS34, IAEA-NO-3, and USGS35), sulfates (IAEA-SO-5, IAEA-SO-6, and NBS 127), or organic material (IAEA-601 benzoic acid, IAEA-602 benzoic acid, and IAEA-600 caffeine), as appropriate to the material being analyzed, had these reference materials been analyzed with unknowns. This procedure ensures that readers will be able to normalize the δ18O values at a later time should it become necessary. The high-temperature reduction technique for analyzing δ18O and δ2H is not as widely applicable as the well-established combustion technique for carbon and nitrogen stable isotope determination. To obtain the most reliable stable isotope data, materials should be treated in an identical fashion; within the same sequence of analyses, samples should be compared with working reference materials that are as similar in nature and in isotopic composition as feasible.

Simplifying and improving the extraction of nitrate from freshwater for stable isotope analyses

Determining the isotopic composition of nitrate (NO3_) in water can prove useful to identify NO3_ sources and to understand its dynamics in aquatic systems. Among the procedures available, the ‘ionexchange resin method’ involves extracting NO3_ from freshwater and converting it into solid silver nitrate (AgNO3), which is then analysed for 15N/14N and 18O/16O ratios. This study describes a simplified methodology where water was not pre-treated to remove dissolved organic carbon (DOC) or barium cations (added to precipitate O-bearing contaminants), which suited samples with high NO3_ ($400 mM or 25 mg L_1 NO3_) and low DOC (typically <417 mM of C or 5 mg L_1 C) levels. % N analysis revealed that a few AgNO3 samples were of low purity (compared with expected % N of 8.2), highlighting the necessity to introduce quality control/quality assurance procedures for silver nitrate prepared from field water samples. Recommendations are then made to monitor % N together with % O (expected at 28.6, i.e. 3.5 fold % N) in AgNO3 in order to better assess the type and gravity of the contamination as well as to identify potentially unreliable data

Mixed-conducting LSC/CGO and Ag/CGO composites for passive oxygen separation membranes

Dense ceramic oxygen separation membranes can pass oxygen perm-selectively at elevated temperature and have potential for improving the performance and reducing the cost of several industrial processes: such as the conversion of natural gas to syngas, or to separate oxygen from air for oxy-fuel combustion in electricity generation (to reduce NOx emissions and facilitate CO2 sequestration). These pressure-driven solid state membranes are based on fast oxygen-ion conducting ceramics, but also need a compensating flow of electrons. Dual-phase composites are attractive since they provide an extra degree of freedom, compared with single phase membranes, for optimising the overall membrane performance. In this study, composites containing gadolinia doped ceria (CGO, Ce0.9Gd0.1O2- ) and either strontium-doped lanthanum cobaltite (LSC, La0.9Sr0.1CoO3- or La0.6Sr0.4CoO3- ) or silver (Ag) were investigated for possible application as oxygen separation membranes in oxy-fuel combustion system. These should combine the high oxygen ion conductivity of CGO with the high electronic conductivity and fast oxygen surface exchange of LSC or silver. Dense mixed-conducting composite materials of LSC/CGO (prepared by powder mixing and sintering) and Ag/CGO composites (prepared by silver plus copper oxide infiltration method) showed high relative density (above 95%), low background gas leakage and also good electrical conduction. The percolation threshold of the electronic conducting component was determined to be approximately 20 vol.% for both LSC compositions and 14 vol.% for Ag. Isotopic exchange and depth profiling by secondary ion mass spectrometry was used to investigated the oxygen tracer diffusion (D*) and surface exchange coefficient (k*) of the composites. Composites just above the electronic percolation threshold exhibited high solid state oxygen diffusivity, fast surface exchange activity moderate thermal expansion and sufficient mechanical strength thus combining the benefits of their constituent materials. The preliminary work on oxygen permeation measurement showed that the reasonable magnitude of oxygen fluxes is possible to be achieved. This indicates that the composites of LSC/CGO and Ag/CGO are promising for further development as passive oxygen separation membranes

Illuminating hydrological processes at the soil-vegetation-atmosphere interface with water stable isotopes

Funded by DFG research project “From Catchments as Organised Systems to Models based on Functional Units” (FOR 1Peer reviewedPublisher PDFPublisher PD

Protein processing characterized by a gel-free proteomics approach

We describe a method for the specific isolation of representative N-terminal peptides of proteins and their proteolytic fragments. Their isolation is based on a gel-free, peptidecentric proteomics approach using the principle of diagonal chromatography. We will indicate that the introduction of an altered chemical property to internal peptides holding a free α-N-terminus results in altered column retention of these peptides, thereby enabling the isolation and further characterization by mass spectrometry of N-terminal peptides. Besides pointing to changes in protein expression levels when performing such proteome surveys in a differential modus, protease specificity and substrate repertoires can be allocated since both are specified by neo-N-termini generated after a protease cleavage event. As such, our gel-free proteomics technology is widely applicable and amenable for a variety of proteome-driven protease degradomics research

A multi-methodological protocol to characterize PDO olive oils

An analytical approach including Panel Test, Isotope Ratio Mass Spectrometry (IRMS) and Nuclear Magnetic Resonance (NMR) spectroscopy was proposed to characterize Italian “Colline Pontine” PDO olive oils (40 samples) of two consecutive crop years. Our approach has evidenced the high quality of these olive oils. Only 6 of 40 olive oils samples were defined as “defective” by the official Panel Test due to the detection of negative sensory attributes. The low variability of isotopic data monitored by IRMS confirmed that the olive oil samples all came from a limited geographical area. NMR spectra did not evidence any chemical composition anomaly in the investigated samples. In order to assess the influence of harvesting year over the olive oil chemical composition, the NMR analysis was extended to other 22 olive oil samples of a third harvesting year. NMR data were submitted to two different statistical methods, namely, analysis of variance (ANOVA) and principal component analysis (PCA) allowing olive oils of three consecutive harvesting years to be grouped

Phase diagram and isotope effect in (PrEu)_0.7Ca_0.3CoO_3 cobaltites exhibiting spin-state transitions

We present the study of magnetization, thermal expansion, specific heat, resistivity, and a.c. susceptibility of (Pr$_{1-y}$Eu$_y$)$_{0.7}$Ca$_{0.3}$CoO$_3$ cobaltites. The measurements were performed on ceramic samples with $y = 0.12 - 0.26$ and $y = 1$. Based on these results, we construct the phase diagram, including magnetic and spin-state transitions. The transition from the low- to intermediate-spin state is observed for the samples with $y > 0.18$, whereas for a lower Eu-doping level, there are no spin-state transitions, but a crossover between the ferromagnetic and paramagnetic states occurs. The effect of oxygen isotope substitution along with Eu doping on the magnetic/spin state is discussed. The oxygen-isotope substitution ($^{16}$O by $^{18}$O) is found to shift both the magnetic and spin-state phase boundaries to lower Eu concentrations. The isotope effect on the spin-state transition temperature ($y > 0.18$) is rather strong, but it is much weaker for the transition to a ferromagnetic state ($y < 0.18$). The ferromagnetic ordering in the low-Eu doped samples is shown to be promoted by the Co$^{4+}$ ions, which favor the formation of the intermediate-spin state of neighboring Co$^{3+}$ ions.Comment: 13 pages, including 11 figures, to be published in Phys. Rev.