Stable Isotope Labeling Approaches Research Articles

Analytical methods for stable isotope labeling to elucidate rapid auxin kinetics in Arabidopsis thaliana.

The phytohormone auxin plays a critical role in plant growth and development. Despite significant progress in elucidating metabolic pathways of the primary bioactive auxin, indole-3-acetic acid (IAA), over the past few decades, key components such as intermediates and enzymes have not been fully characterized, and the dynamic regulation of IAA metabolism in response to environmental signals has not been completely revealed. In this study, we established a protocol employing a highly sensitive liquid chromatography-mass spectrometry (LC-MS) instrumentation and a rapid stable isotope labeling approach. We treated Arabidopsis seedlings with two stable isotope labeled precursors ([13C6]anthranilate and [13C8, 15N1]indole) and monitored the label incorporation into proposed indolic compounds involved in IAA biosynthetic pathways. This Stable Isotope Labeled Kinetics (SILK) method allowed us to trace the turnover rates of IAA pathway precursors and product concurrently with a time scale of seconds to minutes. By measuring the entire pathways over time and using different isotopic tracer techniques, we demonstrated that these methods offer more detailed information about this complex interacting network of IAA biosynthesis, and should prove to be useful for studying auxin metabolic network in vivo in a variety of plant tissues and under different environmental conditions.

Assessing anaerobic microbial degradation rates of crude light oil with reverse stable isotope labelling and community analysis

Oil reservoirs represent extreme environments where anaerobic degradation profoundly influences oil composition and quality. Despite the common observation of biodegraded oil, the microbial degradation rates remain largely unknown. To address this knowledge gap, we conducted microcosm incubations with light oil as carbon source, original formation water and sulfate as electron acceptor, closely mimicking in situ conditions to assess oil degradation rates. Samples were taken from a newly drilled oil well to exclude contamination with injection water and allochthonous microorganisms. At the end of the incubations, microbial community analyses with 16S rRNA gene amplicon sequencing revealed the most prominent phyla as Desulfobacterota, Thermotogota, Bacteroidota, Bacillota (formerly Firmicutes), and Synergistota, collectively accounting for up to 44% of relative abundance. Ion chromatography and reverse stable isotope labeling were used to monitor sulfate reduction and CO2 evolution respectively. We calculated an average degradation rate of 0.35 mmol CO2 per year corresponding to 15.2 mmol CO2/mol CH2(oil) per year. This resembles to approximately 200 years to degrade one gram of oil under the applied, presumably ideal conditions. Factoring in the available oil-water-contact (OWC) zone within the incubations yielded a degradation rate of 120 g CH2 m−2 OWC per year, closely aligning with the modeled degradation rates typically observed in oil reservoirs. Moreover, our study highlighted the utility of the reverse stable isotope labeling (RSIL) approach for measuring complex substrate degradation at minute rates.

Detection and Characterization of Active Microbial Cells in Salt Cavern Brine

Salt caverns have been used for decades as natural gas storage facilities and are now target of large-scale underground H2 storage to secure national energy transition goals. Contrary to CH4, H2 is a universal electron donor for microbial anaerobic respiration. Suitable electron acceptors are sulfate and carbonate, which dissolve from gypsum, anhydrite and lime that can make up 10 % of subsurface salt formations. Whilst sulfate reduction is inherently linked to the formation of H2S, microbial CO2 reduction can generate acetate, which can be used as carbon source by diverse microorganisms. Thus, supporting other microbial side effects, such as H2S formation, clogging and H2 consumption. However, microbial diversity and activity in salt caverns are selectively controlled by salt concentrations close to saturation and limited availability of organic carbon. If these conditions allow for microbial activity was investigated in our study. To circumvent long enrichment times associated with high salinity and limited nutrient availability, we used a stable isotope labelling approach combined with nano-scale secondary ion mass spectrometry analysis (SIP-nanoSIMS) to investigate H2-dependant microbial activity in two brine samples and compared them with that of an extremely halophilic enrichment culture (MP-32). Heavy carbonate and water (13CO2 and 2H2O) served as tracers for microbial activity. Microbial H2 consumption was additionally investigated in microcosm experiments with brine and rock salt over a period of 200 days. Setups with MP-32 served as a positive control. Subsequently, MP-32 was selected for metagenome sequencing to explore potential metabolic pathways and strategies for osmoadaptation. Analysis of the microbial community composition in brine revealed that members of the Desulfohalobiaceae, Halobacteria and Halanaerobiales were present in all caverns and their relative abundance increased during incubation with H2 as electron donor although sulfate reduction was not observed. But incubation with H2 resulted in an increased uptake of 13C from 13CO2 in 1.6 to 3.6 % of the cells compared to incubations without H2. Uptake of 2H from 2H2O was detected in 20 to 30 % of the cells and was higher when H2 was not offered as an electron donor. Similar results were obtained from the enrichment culture MP-32, which was grown in medium with reduced salinity compared to the salt cavern brine. Uptake of 13C was 10-fold higher when incubated with H2 and nearly all cells incorporated 2H with and without H2. A total of eight metagenome-assembled genomes (MAGs) with a completion of more than 90 % could be recovered from MP-32. Two of them belonged to Desulfohalobiaceae and can be characterized as autotrophic sulfate reducers by means of the Acetyl-Coenzyme A pathway that compensate osmotic stress by synthesizing small organic molecules. Collectively, our findings provide a new approach to study microbial activity that is strongly impacted by high salinity and an improved understanding of their genomic potential.

Development of isotopically labelled 65Cu embedded PMMA-65CuO nanocomposites for detection and quantification of PMMA bone cement degradation at trace levels

Polymethyl methacrylate (PMMA) bone cement is a popular bone-filling material for biomedical applications. Bone-cement implant failure is a common occurrence, but the consequence of this failure can be prevented/minimised by early detection of degradation. This work utilises a stable isotope labelling approach to monitor PMMA bone cement degradation. Stable isotopically enriched traceable nanoparticles of 65CuO were synthesized and introduced to PMMA bone cement. Accelerated and normal degradation of 65CuO-loaded PMMA bone cement nanocomposite was performed in acetone and simulated body fluid (SBF). 65Cu was detected and quantified upon PMMA bone cement nanocomposite degradation. The results showed a linear correlation between bone cement degradation and tracer release. When the 65CuO-PMMA composite deteriorated by 6.10 ± 3.04% in acetone, 5.6 ± 2.5 µg of tracer was released in 10 min. Similarly, after one week of degradation in SBF, 0.35 ± 0.31 µg of 65Cu tracer was released, translating to a 0.18 ± 0.09% weight loss. The stable isotopic tracer approach allowed us to measure and detect 65Cu under conditions of high background Cu concentration present in the human body upon 0.2% of PMMA nanocomposite degradation. 65CuO nanoparticles addition to PMMA bone cement did not alter the cytocompatibility (proliferation, differentiation, and osteocalcin formation) in MG-63 cells.

Salt-inducible kinase inhibition promotes the adipocyte thermogenic program and adipose tissue browning

ObjectiveNorepinephrine stimulates the adipose tissue thermogenic program through a β-adrenergic receptor (βAR)–cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling cascade. We discovered that a noncanonical activation of the mechanistic target of rapamycin complex 1 (mTORC1) by PKA is required for the βAR-stimulation of adipose tissue browning. However, the downstream events triggered by PKA-phosphorylated mTORC1 activation that drive this thermogenic response are not well understood. MethodsWe used a proteomic approach of Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) to characterize the global protein phosphorylation profile in brown adipocytes treated with the βAR agonist. We identified salt-inducible kinase 3 (SIK3) as a candidate mTORC1 substrate and further tested the effect of SIK3 deficiency or SIK inhibition on the thermogenic gene expression program in brown adipocytes and in mouse adipose tissue. ResultsSIK3 interacts with RAPTOR, the defining component of the mTORC1 complex, and is phosphorylated at Ser884 in a rapamycin-sensitive manner. Pharmacological SIK inhibition by a pan-SIK inhibitor (HG-9-91-01) in brown adipocytes increases basal Ucp1 gene expression and restores its expression upon blockade of either mTORC1 or PKA. Short-hairpin RNA (shRNA) knockdown of Sik3 augments, while overexpression of SIK3 suppresses, Ucp1 gene expression in brown adipocytes. The regulatory PKA phosphorylation domain of SIK3 is essential for its inhibition. CRISPR-mediated Sik3 deletion in brown adipocytes increases type IIa histone deacetylase (HDAC) activity and enhances the expression of genes involved in thermogenesis such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex protein. We further show that HDAC4 interacts with PGC1α after βAR stimulation and reduces lysine acetylation in PGC1α. Finally, a SIK inhibitor well-tolerated in vivo (YKL-05-099) can stimulate the expression of thermogenesis-related genes and browning of mouse subcutaneous adipose tissue. ConclusionsTaken together, our data reveal that SIK3, with the possible contribution of other SIKs, functions as a phosphorylation switch for β-adrenergic activation to drive the adipose tissue thermogenic program and indicates that more work to understand the role of the SIKs is warranted. Our findings also suggest that maneuvers targeting SIKs could be beneficial for obesity and related cardiometabolic disease.

SolS-catalyzed sulfoxidation of labionin to solabionin drives antibacterial activity of solabiomycins

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) with polar-functionalized fatty acyl groups are newly found lipopeptide-class natural products. We recently employed a combined approach of genome mining and stable isotope labeling and discovered solabiomycins as one of the polar-functionalized fatty-acylated RiPPs (PFARs) from Streptomyces lydicus NBRC13058. The solabiomycins contained a characteristic sulfoxide group in the labionin moiety referred to as the ‘solabionin’ structure for the RiPP moiety. A previous gene knockout experiment indicated that solS, which encodes a putative flavin adenine dinucleotide (FAD)-nicotinamide adenine dinucleotide (phosphate) (NAD(P))-binding protein, is involved in the sulfoxidation of an alkyl sulfide in the solabionin. In this study, we isolated deoxysolabiomycins A and B from ΔsolS mutant and fully determined the chemical structures using a series of NMR experiments. We also tested the bioactivity of deoxysolabiomycins against Gram-positive bacteria, including Mycolicibacterium smegmatis, and notably found that the sulfoxide is critical for the antibacterial activity. To characterize the catalytic activity of SolS, the recombinant protein was incubated with a putative substrate, deoxysolabiomycins, and the cofactors FAD and NADPH. In vitro reactions demonstrated that SolS catalyzes the sulfoxidation, converting deoxysolabiomycins to solabiomycins.

Protein kinetics of superoxide dismutase-1 in familial and sporadic amyotrophic lateral sclerosis.

Accumulation of misfolded superoxide dismutase-1 (SOD1) is a pathological hallmark of SOD1-related amyotrophic lateral sclerosis (ALS) and is observed in sporadic ALS where its role in pathogenesis is controversial. Understanding in vivo protein kinetics may clarify how SOD1 influences neurodegeneration and inform optimal dosing for therapies that lower SOD1 transcripts. We employed stable isotope labeling paired with mass spectrometry to evaluate in vivo protein kinetics and concentration of soluble SOD1 in cerebrospinal fluid (CSF) of SOD1 mutation carriers, sporadic ALS participants and controls. A deaminated SOD1 peptide, SDGPVKV, that correlates with protein stability was also measured. In participants with heterozygous SOD1A5V mutations, known to cause rapidly progressive ALS, mutant SOD1 protein exhibited ~twofold faster turnover and ~ 16-fold lower concentration compared to wild-type SOD1 protein. SDGPVKV levels were increased in SOD1A5V carriers relative to controls. Thus, SOD1 mutations impact protein kinetics and stability. We applied this approach to sporadic ALS participants and found that SOD1 turnover, concentration, and SDGPVKV levels are not significantly different compared to controls. These results highlight the ability of stable isotope labeling approaches and peptide deamidation to discern the influence of disease mutations on protein kinetics and stability and support implementation of this method to optimize clinical trial design of gene and molecular therapies for neurological disorders. Clinicaltrials.gov: NCT03449212.

Investigation of the Quinone-quinone and Quinone-catechol products using 13C labeling, UPLC-Q-TOF/MS and UPLC-Q-Exactive Orbitrap/MS

Quinones are highly reactive oxidants and play an essential role in inducing quality deterioration of fruit and vegetable products. Here, a novel stable isotope-labeling approach in combination with high-resolution tandem mass spectrometry UPLC-Q-TOF/MS and UPLC-Q-Exactive Orbitrap/MS, was successfully applied in tracking quinone reaction pathways in both real wines and model reaction systems. Unexpectedly, the binding products of quinone-quinone and quinone-catechol that are not derived from either nucleophilic reaction or redox reaction were discovered and showed the significant high peak area.Self-coupling reactions of semiquinone radicals might provide a possible interpretation for the formation of quinone-quinone products, and a charge transfer reaction coupled with a complementary donor–acceptor interaction is feasibly responsible for the products with a quinone-catechol structure. These findings endow a new perspective for quinone metabolic pathway in foods.

A peroxisomal heterodimeric enzyme is involved in benzaldehyde synthesis in plants

Benzaldehyde, the simplest aromatic aldehyde, is one of the most wide-spread volatiles that serves as a pollinator attractant, flavor, and antifungal compound. However, the enzyme responsible for its formation in plants remains unknown. Using a combination of in vivo stable isotope labeling, classical biochemical, proteomics and genetic approaches, we show that in petunia benzaldehyde is synthesized via the β-oxidative pathway in peroxisomes by a heterodimeric enzyme consisting of α and β subunits, which belong to the NAD(P)-binding Rossmann-fold superfamily. Both subunits are alone catalytically inactive but, when mixed in equal amounts, form an active enzyme, which exhibits strict substrate specificity towards benzoyl-CoA and uses NADPH as a cofactor. Alpha subunits can form functional heterodimers with phylogenetically distant β subunits, but not all β subunits partner with α subunits, at least in Arabidopsis. Analysis of spatial, developmental and rhythmic expression of genes encoding α and β subunits revealed that expression of the gene for the α subunit likely plays a key role in regulating benzaldehyde biosynthesis.

Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells.

For decades, “all-or-none” and “kiss-and-run” were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I–, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I– in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically.

Trophic upgrading of long-chain polyunsaturated fatty acids by polychaetes: a stable isotope approach using Alitta virens

Polychaete worms are rich sources of polyunsaturated fatty acids (PUFA) and are increasingly incorporated into aquaculture broodstock diets. Conventionally, the build-up of PUFA in polychaetes was considered passive, with direct accumulation along the food web, originating with microalgae and other primary producers. However, it has been argued that polychaetes (and other multicellular eukaryotes) are capable of PUFA biosynthesis through the elongation and desaturation of precursor lipids. We further test this hypothesis in the ecologically and economically important nereid polychaete Alitta virens by adopting a stable isotope labelling approach. Worms were fed a 13C-1-palmitic acid (C16:0) enriched diet with the resulting isotopically enriched lipid products identified over a 7-day period. The data showed strong evidence of lipid elongation and desaturation, but with a high rate of PUFA turnover. A putative biosynthetic pathway is proposed, terminating with docosahexaenoic acid (DHA) via arachidonic (AA) and eicosapentaenoic acids (EPA) and involving a Δ8 desaturase.

Characterization of the PRODUCTION of ANTHOCYANIN PIGMENT 1 Arabidopsis dominant mutant using DLEMMA dual isotope labeling approach

Stable isotope labeling has emerged as a valuable tool for metabolite identification and quantification. In this study, we employed DLEMMA, a dual stable isotope labeling approach to identify and track phenylpropanoid pathway in Arabidopsis thaliana. Three forms of phenylalanine (Phe), including unlabeled, Phe13C6 and Phe13C62H5, were used as feeding precursors. The unique isotopic pattern obtained from MS spectra significantly simplified data processing and facilitated global mining of Phe-derived metabolites. Following this approach, we have identified 35 phenylalanine-derived metabolites with high confidence. We next compared phenylpropanoids contents between leaves of wild type (WT) and the dominant PRODUCTION OF ANTHOCYANIN PIGMENT 1 (pap1-D) Arabidopsis thaliana mutant using a combined sample matrices and label-swap approach. This approach was designed to correct any unequal matrix effects between the two divergent samples, and any possible uneven label incorporation efficiency between the two differently labeled Phe precursors. Thirty of the 35 identified metabolites were found differential between WT and pap1-D leaves. Our results shown that the ectopic PAP1 expression led to significant accumulation of cyanidin-type anthocyanins, quercetin-type flavonols and hydroxycinnamic acids and their glycosylated derivatives. While levels of kaempferol glycosides and a hydroxycinnamic acid amide were reduced in the pap1-D leaves.

Linkage-selective derivatization for glycosylation site- and glycoform-specific characterization of sialic acid isomers using mass spectrometry.

Here, we developed a linkage-selective derivatization approach for the differentiation and relative quantification of α-2,3- and α-2,6-linked sialic acids in a site- and glycoform-specific manner. Linkage-selective derivatization with isotope molecules discriminates the isomeric glycopeptides easily using MS and provided a tool for biomarker discovery using the quantitative analysis of isomeric glycopeptides.

Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10μM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.

Photoprotective Acclimation of the Arabidopsis thaliana Leaf Proteome to Fluctuating Light.

Plants are subjected to strong fluctuations in light intensity in their natural growth environment, caused both by unpredictable changes due to weather conditions and movement of clouds and upper canopy leaves and predictable changes during day-night cycle. The mechanisms of long-term acclimation to fluctuating light (FL) are still not well understood. Here, we used quantitative mass spectrometry to investigate long-term acclimation of low light-grown Arabidopsis thaliana to a FL condition that induces mild photooxidative stress. On the third day of exposure to FL, young and mature leaves were harvested in the morning and at the end of day for proteome analysis using a stable isotope labeling approach. We identified 2,313 proteins, out of which 559 proteins exhibited significant changes in abundance in at least one of the four experimental groups (morning-young, morning-mature, end-of-day-young, end-of-day-mature). A core set of 49 proteins showed significant responses to FL in three or four experimental groups, which included enhanced accumulation of proteins involved in photoprotection, cyclic electron flow around photosystem I, photorespiration, and glycolysis, while specific glutathione transferases and proteins involved in translation and chlorophyll biosynthesis were reduced in abundance. In addition, we observed pathway- and protein-specific changes predominantly at the end of day, whereas few changes were observed exclusively in the morning. Comparison of the proteome data with the matching transcript data revealed gene- and protein-specific responses, with several chloroplast-localized proteins decreasing in abundance despite increased gene expression under FL. Together, our data shows moderate but widespread alterations of protein abundance during acclimation to FL and suggests an important role of post-transcriptional regulation of protein abundance.

Inorganic Nitrate Promotes Glucose Uptake and Oxidative Catabolism in White Adipose Tissue Through the XOR-Catalyzed Nitric Oxide Pathway.

An aging global population combined with sedentary lifestyles and unhealthy diets has contributed to an increasing incidence of obesity and type 2 diabetes. These metabolic disorders are associated with perturbations to nitric oxide (NO) signaling and impaired glucose metabolism. Dietary inorganic nitrate, found in high concentration in green leafy vegetables, can be converted to NO in vivo and demonstrates antidiabetic and antiobesity properties in rodents. Alongside tissues including skeletal muscle and liver, white adipose tissue is also an important physiological site of glucose disposal. However, the distinct molecular mechanisms governing the effect of nitrate on adipose tissue glucose metabolism and the contribution of this tissue to the glucose-tolerant phenotype remain to be determined. Using a metabolomic and stable-isotope labeling approach, combined with transcriptional analysis, we found that nitrate increases glucose uptake and oxidative catabolism in primary adipocytes and white adipose tissue of nitrate-treated rats. Mechanistically, we determined that nitrate induces these phenotypic changes in primary adipocytes through the xanthine oxidoreductase-catalyzed reduction of nitrate to NO and independently of peroxisome proliferator-activated receptor-α. The nitrate-mediated enhancement of glucose uptake and catabolism in white adipose tissue may be a key contributor to the antidiabetic effects of this anion.

Using stable isotope labeling approach and two dimensional correlation spectroscopy to explore the turnover cycles of different carbon structures in extracellular polymeric substances

Extracellular polymeric substances (EPS) from activated sludge comprise many organic constituents with polysaccharides and proteins as the main components of two different functionalities. Despite a number of previous EPS studies, a fundamental question remained unanswered, namely, whether the different EPS components would have the same turnover cycle (i.e., formation/dissolution) in biological wastewater treatment systems. In this study, we employed a stable isotope labeling approach based on isotope-enriched substrates (i.e., 13C-glucose and 15NH4Cl) to examine the potential discrepancies in the turnover cycles among different major EPS constituents. Our results, based on substrate consumption in a batch bioreactor, evidenced the existence of differences in carbon and nitrogen cycles within bulk EPS with an earlier replenishment of organic carbon relative to organic nitrogen. The changes in the 13C nuclear magnetic resonance (13C NMR) spectra of EPS with operation clarified the relative differences in the turnover periods among several identified EPS structures with different chemical functionalities. Two-dimensional correlation spectroscopy (2D-COS) on the 13C NMR spectra further showed that the substrate-assimilated carbon functional groups appear to preferably formed within bulk EPS in the order of O-alkyl carbons > amides > α amino acids > aliphatic carbons. This study provides a novel insight into the dissimilar formation rates of different EPS structures after substrate assimilation. This isotope labeling approach can be further applied to determine the mass balance among the substrate, biomass, and bound/soluble EPS within activated sludge systems.

Proteomic analysis of haptenation by skin sensitisers: Diphencyprone and ethyl acrylate

The potential risk of skin sensitisation, associated with the development of allergic contact dermatitis (ACD), is a consideration in the safety assessment of new ingredients for use in personal care products. Protein haptenation in skin by sensitising chemicals is the molecular initiating event causative of skin sensitisation. Current methods for monitoring skin sensitisation rely on limited reactivity assays, motivating interest in the development of proteomic approaches to characterise the skin haptenome. Increasing our mechanistic understanding of skin sensitisation and ACD using proteomics presents an opportunity to develop non-animal predictive methods and/or risk assessment approaches. Previously, we have used a novel stable isotope labelling approach combined with data independent mass spectrometry (HDMSE) to characterise the haptenome for a number of well-known sensitisers. We have now extended this work by characterising the haptenome of the sensitisers Diphenylcyclopropenone (DPCP) and Ethyl Acrylate (EA) with the model protein Human Serum Albumin (HSA) and the complex lysates of the skin keratinocyte, HaCaT cell line. We show that haptenation in complex nucleophilic models is not random, but a specific, low level and reproducible event. Proteomic analysis extends our understanding of sensitiser reactivity beyond simple reactivity assays and offers a route to monitoring haptenation in living cells.

Unexpected role of canonical aerobic methanotrophs in upland agricultural soils

Aerobic oxidation of methane at (circum-)atmospheric concentrations (<40 ppmv) has long been assumed to be catalyzed by the as-yet-uncultured high-affinity methanotrophs in well-aerated, non-wetland (upland) soils, the only known biological methane sink globally. Although the low-affinity canonical methanotrophs with cultured representatives have been detected along with the high-affinity ones, their role as a methane sink in upland soils remains enigmatic. Here, we show that canonical methanotrophs can contribute to (circum-)atmospheric methane uptake in agricultural soils. We performed a stable-isotope 13CCH4 labelling incubation in the presence and absence of bio-based residues that were added to the soil to track the flow of methane. Residue amendment transiently stimulated methane uptake rate (<50 days). Soil methane uptake was sustained throughout the incubation (130 days), concomitant to the enrichment of 13CCO2. The 13C-enriched phospholipid fatty acids (PLFAs) were distinct in both soils, irrespective of amendments, and were unambiguously assigned almost exclusively to canonical alphaproteobacterial methanotrophs with cultured representatives. 16S rRNA and pmoA gene sequence analyses revealed that the as-yet-uncultured high-affinity methanotrophs were virtually absent in these soils. The stable-isotope labelling approach allowed to attribute soil methane uptake to canonical methanotrophs, whereas these were not expected to consume (circum-)atmospheric methane. Our findings thus revealed an overlooked reservoir of high-affinity methane-oxidizers represented by the canonical methanotrophs in agriculture-impacted upland soils. Given that upland agricultural soils have been thought to marginally or do not contribute to atmospheric methane consumption due to the vulnerability of the high-affinity methanotrophs, our findings suggest a thorough revisiting of the contribution of agricultural soils, and the role of agricultural management to mitigation of climate change.

Tracing oxidation reaction pathways in wine using 13C isotopolog patterns and a putative compound database

Quinones are key reactive electrophilic intermediates that are formed in abundance during wine oxidation and have a significant impact on wine character, altering color and flavor. They readily react with nucleophiles, such as SO2 and glutathione. Some nucleophiles have been reported to react with quinones in model wines, but many of the corresponding products have not been confirmed in real wines. Here, a stable isotope labeling approach employing 13C6 labeled ortho-quinone was evaluated in combination with an exact-mass list of putative products. The measurements were taken using high performance liquid chromatography coupled to time-of-flight mass spectrometry. Fourteen compounds were detected based on a match in the table plus an M+6 mass isotope pattern derived from the labeled quinone mixture. The presence of the mass label also establishes reaction pathways for the formation of these compounds, and suggests how wine catechols could react during wine oxidation, demonstrating the insight provided by this analytical technique.