ABSTRACT
The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.
Subject(s)
Bile Acids and Salts , Gastrointestinal Microbiome , Metabolomics , Tandem Mass Spectrometry , Animals , Humans , Bile Acids and Salts/chemistry , Metabolomics/methods , Polyamines , Tandem Mass Spectrometry/methods , Databases, ChemicalABSTRACT
Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.
Subject(s)
Cyanobacteria , Cyanobacteria/metabolism , Cyanobacteria/chemistry , Cyanobacteria/genetics , Humans , Multigene Family , Cell Line, Tumor , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/metabolismABSTRACT
SUMMARY: Untargeted metabolomics data analysis is highly labour intensive and can be severely frustrated by both experimental noise and redundant features. Homologous polymer series is a particular case of features that can either represent large numbers of noise features or alternatively represent features of interest with large peak redundancy. Here, we present homologueDiscoverer, an R package that allows for the targeted and untargeted detection of homologue series as well as their evaluation and management using interactive plots and simple local database functionalities. AVAILABILITY AND IMPLEMENTATION: homologueDiscoverer is freely available at GitHub https://github.com/kevinmildau/homologueDiscoverer. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Software , Tandem Mass Spectrometry , Chromatography, Liquid , Metabolomics , Data AnalysisABSTRACT
When performing chromatography-mass spectrometry-based nontargeted metabolomics, or exposomics, one of the key steps in the analysis is to obtain MS1-based feature tables. Inapt parameter settings in feature detection will result in missing or wrong quantitative values and might ultimately lead to downstream incorrect biological interpretations. However, until recently, no strategies to assess the completeness and abundance accuracy of feature tables were available. Here, we show that mzRAPP enables the generation of benchmark peak lists by using an internal set of known molecules in the analyzed data set. Using the benchmark, the completeness and abundance accuracy of feature tables can be assessed in an automated pipeline. We demonstrate that our approach adds to other commonly applied quality assurance methods such as manual or automatized parameter optimization techniques or removal of false-positive signals. Moreover, we show that as few as 10 benchmark molecules can already allow for representative performance metrics to further improve quantitative biological understanding.
Subject(s)
Metabolomics , Chromatography, Liquid/methods , Mass Spectrometry/methods , Metabolomics/methodsABSTRACT
SUMMARY: Reliability assessment of automated pre-processing of liquid chromatography-high resolution mass spectrometry data presents a significant challenge. Here, we present a tool named mzRAPP, which generates and validates a benchmark from user-supplied information and later utilizes it for reliability assessment of data pre-processing. As a result, mzRAPP produces several performance metrics for different steps of the pre-processing workflow, supporting five of the most commonly used pre-processing tools. AVAILABILITY AND IMPLEMENTATION: mzRAPP is implemented in R and can be downloaded from GitHub under GNU GPL v.3.0 licence. Extensive documentation, background and examples are available at (https://github.com/YasinEl/mzRAPP). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
ABSTRACT
We propose a fully automated novel workflow for lipidomics based on flow injection, followed by liquid chromatography-high-resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed-phase LC-HRMS with absolute quantification by using a large number of lipid species-specific and/or retention time (RT)-matched/class-specific calibrants. The lipidome of 13C-labelled yeast (LILY) provided a large panel of cost-effective internal standards (ISTDs) covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). The workflow in combination with the LILY lipid panel enables simultaneous quantification via (1) external multi-point calibration with internal standardization and (2) internal one-point calibration with LILY as a surrogate ISTD, increasing the coverage while keeping the accuracy and throughput high. Extensive measures on quality control allowed us to rank the calibration strategies and to automatically select the calibration strategy of the highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis, with a limit of detection in the low femtomolar range, and provided validation tools together with absolute concentration values for >350 lipids in human plasma on a species level. Based on the selected standard panel, lipids from 7 classes (LPC, LPE, PC, PE, PI, DG, TG) passed stringent quality filters, which included QC accuracy, a precision and recovery bias of <30% and concentrations within the 99% confidence interval of the international laboratory comparison of SRM 1950, NIST, USA. The quantitative values are independent of common deuterated or non-endogenous ISTDs, thus offering cross-validation of different lipid methods and further standardizing lipidomics.
ABSTRACT
13C metabolite tracer and metabolic flux analyses require upfront experimental planning and validation tools. Here, we present a validation scheme including a comparison of different LC methods that allow for customization of analytical strategies for tracer studies with regard to the targeted metabolites. As the measurement of significant changes in labeling patterns depends on the spectral accuracy, we investigate this aspect comprehensively for high-resolution orbitrap mass spectrometry combined with reversed-phase chromatography, hydrophilic interaction liquid chromatography, or anion-exchange chromatography. Moreover, we propose a quality control protocol based on (1) a metabolite containing selenium to assess the instrument performance and on (2) in vivo synthesized isotopically enriched Pichia pastoris to validate the accuracy of carbon isotopologue distributions (CIDs), in this case considering each isotopologue of a targeted metabolite panel. Finally, validation involved a thorough assessment of procedural blanks and matrix interferences. We compared the analytical figures of merit regarding CID determination for over 40 metabolites between the three methods. Excellent precisions of less than 1% and trueness bias as small as 0.01-1% were found for the majority of compounds, whereas the CID determination of a small fraction was affected by contaminants. For most compounds, changes of labeling pattern as low as 1% could be measured. Graphical abstract.
Subject(s)
Carbon Isotopes/analysis , Chromatography, Ion Exchange/methods , Chromatography, Reverse-Phase/methods , Mass Spectrometry/methods , Validation Studies as Topic , Anion Exchange Resins/chemistry , Carbon Isotopes/standards , Hydrophobic and Hydrophilic Interactions , Pichia/chemistry , Reference Standards , Selenium/chemistryABSTRACT
Lipid identification and quantification are essential objectives in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity, and their dynamic range. In this work, we developed a tailored method for profiling and quantification combining (1) isotope dilution, (2) enhanced isomer separation by C30 fused-core reversed-phase material, and (3) parallel Orbitrap and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer. The combination of parallelizable ion analysis without time loss together with different fragmentation techniques (HCD/CID) and an inclusion list led to higher quality in lipid identifications exemplified in human plasma and yeast samples. Moreover, we used lipidome isotope-labeling of yeast (LILY)-a fast and efficient in vivo labeling strategy in Pichia pastoris-to produce (nonradioactive) isotopically labeled eukaryotic lipid standards in yeast. We integrated the 13C lipids in the LC-MS workflow to enable relative and absolute compound-specific quantification in yeast and human plasma samples by isotope dilution. Label-free and compound-specific quantification was validated by comparison against a recent international interlaboratory study on human plasma SRM 1950. In this way, we were able to prove that LILY enabled quantification leads to accurate results, even in complex matrices. Excellent analytical figures of merit with enhanced trueness, precision and linearity over 4-5 orders of magnitude were observed applying compound-specific quantification with 13C-labeled lipids. We strongly believe that lipidomics studies will benefit from incorporating isotope dilution and LC-MSn strategies.
Subject(s)
Chromatography, Reverse-Phase/methods , Lipids/blood , Mass Spectrometry/methods , Carbon Isotopes/analysis , Carbon Isotopes/blood , Humans , Indicator Dilution Techniques , Lipids/analysis , Workflow , Yeasts/chemistryABSTRACT
A novel integrated metabolomics/lipidomics workflow is introduced enabling high coverage of polar metabolites and non-polar lipids within one analytical run. Dual HILIC and RP chromatography were combined to high-resolution mass spectrometry. As a major advantage, only one data file per sample was obtained by fully automated simultaneous analysis of two extracts per sample. Hence, the unprecedented high coverage without compromise on analytical throughput was not only obtained by the orthogonality of the chromatographic separations, but also by the implementation of dedicated sample preparation procedures resulting in optimum extraction efficiency for both sub-omes. Thus, the method addressed completely hydrophilic sugars and organic acids next to water-insoluble triglycerides. As for the timing of the dual chromatography setup, HILIC and RP separation were performed consecutively. However, re-equilibration of the HILIC column during elution of RP compounds and vice versa reduced the overall analysis time by one third to 32 min. Application to the Standard Reference Material SRM 1950 - Metabolites in Frozen Human Plasma resulted in >100 metabolite and >380 lipid identifications based on accurate mass implementing fast polarity switching and acquiring data dependent MS2 spectra with the use of automated exclusion lists. Targeted quantification based on external calibrations and 13C labeled yeast internal standards was successfully accomplished for 59 metabolites. Moreover, the potential for lipid quantification was shown integrating non-endogenous lipids as internal standards. In human plasma, concentrations ranging over 4 orders of magnitude (low nM to high µM) were assessed.
Subject(s)
Chromatography, Liquid/methods , Chromatography, Reverse-Phase/methods , Lipids/blood , Metabolomics/methods , Humans , Hydrophobic and Hydrophilic Interactions , Mass Spectrometry/methodsABSTRACT
Given the chemical diversity of lipids and their biological relevance, suitable methods for lipid profiling and quantification are demanded to reduce sample complexity and analysis times. In this work, we present a novel on-line chromatographic method coupling hydrophilic interaction liquid chromatography (HILIC) dedicated to class-specific separation of polar lipid to reversed-phase chromatography (RP) for non-polar lipid analysis. More specifically, the void volume of the HILIC separation-consisting of non-polar lipids- is transferred to the orthogonal RP column enabling the on-line combination of HILIC with RP without any dilution in the second dimension. In this setup the orthogonal HILIC and RP separations were performed in parallel and the effluents of both columns were combined prior to high-resolution MS detection, offering the full separation space in one analytical run. Rapid separation for both polar and non-polar lipids within only 15 min (including reequilibration time) was enabled using sub-2 µm particles and UHPLC. The method proved to be robust with excellent retention time stability (RSDs < 1%) and LODs in the fmol to pmol (absolute on column) range even in the presence of complex biological matrix such as human plasma. The presented high-resolution LC-MS/MS method leads to class-specific separation of polar lipids and separation of non-polar lipids which is lost in conventional HILIC separations. HILIC-RP-MS is a promising tool for targeted and untargeted lipidomics workflows as three interesting features are combined namely (1) the decreased run time of state of the art shotgun MS methods, (2) the elevated linear dynamic range inherent to chromatographic separation and (3) increased level of identification by separation of polar and non-polar lipid classes.
ABSTRACT
Bile acids regulate nutrient absorption and mitochondrial function, they establish and maintain gut microbial community composition and mediate inflammation, and they serve as signalling molecules that regulate appetite and energy homeostasis. The observation that there are hundreds of bile acids, especially many amidated bile acids, necessitates a revision of many of the classical descriptions of bile acids and bile acid enzyme functions. For example, bile salt hydrolases also have transferase activity. There are now hundreds of known modifications to bile acids and thousands of bile acid-associated genes, especially when including the microbiome, distributed throughout the human body (for example, there are >2,400 bile salt hydrolases alone). The fact that so much of our genetic and small-molecule repertoire, in both amount and diversity, is dedicated to bile acid function highlights the centrality of bile acids as key regulators of metabolism and immune homeostasis, which is, in large part, communicated via the gut microbiome.
Subject(s)
Bile Acids and Salts , Bile Acids and Salts/metabolism , Humans , Gastrointestinal Microbiome/physiology , Homeostasis/physiologyABSTRACT
The consumption of alcohol and caffeine affects the lives of billions of individuals worldwide. Although recent evidence indicates that caffeine impairs the reinforcing properties of alcohol, a characterization of its effects on alcohol-stimulated mesolimbic dopamine (DA) function was lacking. Acting as the pro-drug of salsolinol, alcohol excites DA neurons in the posterior ventral tegmental area (pVTA) and increases DA release in the nucleus accumbens shell (AcbSh). Here we show that caffeine, via antagonistic activity on A2A adenosine receptors (A2AR), prevents alcohol-dependent activation of mesolimbic DA function as assessed, in-vivo, by brain microdialysis of AcbSh DA and, in-vitro, by electrophysiological recordings of pVTA DA neuronal firing. Accordingly, while the A1R antagonist DPCPX fails to prevent the effects of alcohol on DA function, both caffeine and the A2AR antagonist SCH 58261 prevent alcohol-dependent pVTA generation of salsolinol and increase in AcbSh DA in-vivo, as well as alcohol-dependent excitation of pVTA DA neurons in-vitro. However, caffeine also prevents direct salsolinol- and morphine-stimulated DA function, suggesting that it can exert these inhibitory effects also independently from affecting alcohol-induced salsolinol formation or bioavailability. Finally, untargeted metabolomics of the pVTA showcases that caffeine antagonizes alcohol-mediated effects on molecules (e.g. phosphatidylcholines, fatty amides, carnitines) involved in lipid signaling and energy metabolism, which could represent an additional salsolinol-independent mechanism of caffeine in impairing alcohol-mediated stimulation of mesolimbic DA transmission. In conclusion, the outcomes of this study strengthen the potential of caffeine, as well as of A2AR antagonists, for future development of preventive/therapeutic strategies for alcohol use disorder.
ABSTRACT
microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health.
Subject(s)
Metabolomics , Tandem Mass Spectrometry , Humans , Metabolomics/methods , Databases, FactualABSTRACT
Covering a wide spectrum of molecules is essential for global metabolome assessment. While metabolomics assays are most frequently carried out in microbore LC-MS analysis, reducing the size of the analytical platform has proven its ability to boost sensitivity for specific -omics applications. In this study, we elaborate the impact of LC miniaturization on exploratory small-molecule LC-MS analysis, focusing on chromatographic properties with critical impact on peak picking and statistical analysis. We have assessed a panel of small molecules comprising endogenous metabolites and environmental contaminants covering three flow regimes-analytical, micro-, and nano-flow. Miniaturization to the micro-flow regime yields moderately increased sensitivity as compared to the nano setup, where median sensitivity gains around 80-fold are observed in protein-precipitated blood plasma extract. This gain resulting in higher coverage at low µg/L concentrations is compound dependent. At the same time, the nano-LC-high-resolution mass spectrometry (HRMS) approach reduces the investigated chemical space as a consequence of the trap-and-elute nano-LC platform. Finally, while all three setups show excellent retention time stabilities, rapid gradients jeopardize the peak area repeatability of the nano-LC setup. Micro-LC offers the best compromise between improving signal intensity and metabolome coverage, despite the fact that only incremental gains can be achieved. Hence, we recommend using micro-LC for wide-target small-molecule trace bioanalysis and global metabolomics of abundant samples.
ABSTRACT
Covalent or non-covalent heterogeneous multimerization of molecules associated with extracts from biological samples analyzed via LC-MS are quite difficult to recognize/annotate and therefore the prevalence of multimerization remains largely unknown. In this study, we utilized 13C labeled and unlabeled Pichia pastoris extracts to recognize heterogeneous multimers. More specifically, between 0.8% and 1.5% of the biologically-derived features detected in our experiments were confirmed to be heteromers, about half of which we could successfully annotate with monomeric partners. Interestingly, we found specific chemical classes such as nucleotides to disproportionately contribute to heteroadducts. Furthermore, we compiled these compounds into the first MS/MS library that included data from heteromultimers to provide a starting point for other labs to improve the annotation of such ions in other metabolomics data sets. Then, the detected heteromers were also searched in publicly accessible LC-MS datasets available in Metabolights, Metabolomics WB and GNPS/MassIVE to demonstrate that these newly annotated ions are also relevant to other public datasets. Furthermore, in additional datasets (Triticum aestivum, Fusarium graminearum, and Trichoderma reesei) our developed workflow also detected 0.5%-4.9% of metabolite features to originate from heterodimers, demonstrating heteroadducts to be present in metabolomics studies at a low percentage.
Subject(s)
Metabolomics , Tandem Mass Spectrometry , Chromatography, Liquid , Ions/chemistry , NucleotidesABSTRACT
Gangliosides are an indispensable glycolipid class concentrated on cell surfaces with a critical role in stem cell differentiation. Nonetheless, owing to the lack of suitable methods for scalable analysis covering the full scope of ganglioside molecular diversity, their mechanistic properties in signaling and differentiation remain undiscovered to a large extent. This work introduces a sensitive and comprehensive ganglioside assay based on liquid chromatography, high-resolution mass spectrometry, and multistage fragmentation. Complemented by an open-source data evaluation workflow, we provide automated in-depth lipid species-level and molecular species-level annotation based on decision rule sets for all major ganglioside classes. Compared to conventional state-of-the-art methods, the presented ganglioside assay offers (1) increased sensitivity, (2) superior structural elucidation, and (3) the possibility to detect novel ganglioside species. A major reason for the highly improved sensitivity is the optimized spectral readout based on the unique capability of two parallelizable mass analyzers for multistage fragmentation. We demonstrated the high-throughput universal capability of our novel analytical strategy by identifying 254 ganglioside species. As a proof of concept, 137 unique gangliosides were annotated in native and differentiated human mesenchymal stem cells including 78 potential cell-state-specific markers and 38 previously unreported gangliosides. A general increase of the ganglioside numbers upon differentiation was observed as well as cell-state-specific clustering based on the ganglioside species patterns. The combination of the developed glycolipidomics assay with the extended automated annotation tool enables comprehensive in-depth ganglioside characterization as shown on biological samples of interest. Our results suggest ganglioside patterns as a promising quality control tool for stem cells and their differentiation products. Additionally, we believe that our analytical workflow paves the way for probing glycolipid-based biochemical processes shedding light on the enigmatic processes of gangliosides and glycolipids in general.
ABSTRACT
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.
ABSTRACT
Oxidative stress and reactive oxygen species (ROS) are central to many physiological and pathophysiological processes. However, due to multiple technical challenges, it is hard to capture a comprehensive readout of the cell, involving both biochemical and functional status. We addressed this problem by developing a fully parallelized workflow for metabolomics (providing absolute quantities for > 100 metabolites including TCA cycle, pentose phosphate pathway, purine metabolism, glutathione metabolism, cysteine and methionine metabolism, glycolysis and gluconeogenesis) and live cell imaging microscopy. The correlative imaging strategy was applied to study morphological and metabolic adaptation of cancer cells upon short-term hydrogen peroxide (H2O2) exposure in vitro. The combination provided rich metabolic information at the endpoint of exposure together with imaging of mitochondrial effects. As a response, superoxide concentrations were elevated with a strong mitochondrial localization, and multi-parametric image analysis revealed a shift towards fragmentation. In line with this, metabolism reflected both the impaired mitochondrial function and shifts to support the first-line cellular defense and compensate for energy loss. The presented workflow combining high-end technologies demonstrates the applicability for the study of short-term oxidative stress, but it can be suitable for the in-depth study of various short-term oxidative and other cellular stress-related phenomena.
ABSTRACT
Resistance development is a major obstacle for platinum-based chemotherapy, with the anticancer drug oxaliplatin being no exception. Acquired resistance is often associated with altered drug accumulation. In this work we introduce a novel -omics workflow enabling the parallel study of platinum drug uptake and its distribution between nucleus/protein and small molecule fraction along with metabolic changes after different treatment time points. This integrated metallomics/metabolomics approach is facilitated by a tailored sample preparation workflow suitable for preclinical studies on adherent cancer cell models. Inductively coupled plasma mass spectrometry monitors the platinum drug, while the metabolomics tool-set is provided by hydrophilic interaction liquid chromatography combined with high-resolution Orbitrap mass spectrometry. The implemented method covers biochemical key pathways of cancer cell metabolism as shown by a panel of >130 metabolite standards. Furthermore, the addition of yeast-based 13C-enriched internal standards upon extraction enabled a novel targeted/untargeted analysis strategy. In this study we used our method to compare an oxaliplatin sensitive human colon cancer cell line (HCT116) and its corresponding resistant model. In the acquired oxaliplatin resistant cells distinct differences in oxaliplatin accumulation correlated with differences in metabolomic rearrangements. Using this multi-omics approach for platinum-treated samples facilitates the generation of novel hypotheses regarding the susceptibility and resistance towards oxaliplatin.
Subject(s)
Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Oxaliplatin/pharmacology , Antineoplastic Agents/pharmacokinetics , Chromatography, Liquid/methods , Colonic Neoplasms/metabolism , Drug Resistance, Neoplasm , HCT116 Cells , Humans , Mass Spectrometry/methods , Metabolomics/methods , Oxaliplatin/pharmacokineticsABSTRACT
Iron is a micronutrient of particular interest as low levels of iron limit primary production of phytoplankton and carbon fluxes in extended regions of the world's oceans. Sphagnum-peatland runoff is extraordinarily rich in dissolved humic-bound iron. Given that several of the world's largest wetlands are Sphagnum-dominated peatlands, this ecosystem type may serve as one of the major sources of iron to the ocean. Here, we studied five near-coastal creeks in North Scotland using freshwater/seawater mixing experiments of natural creek water and synthetic seawater based on a (59)Fe radiotracer technique combined with isotopic characterization of dissolved organic carbon by Accelerator Mass Spectrometry. Three of the creeks meander through healthy Sphagnum-dominated peat bogs and the two others through modified peatlands which have been subject to artificial drainage for centuries. The results revealed that, at the time of sampling (August 16-24, 2014), the creeks that run through modified peatlands delivered 11-15µg iron per liter creek water to seawater, whereas the creeks that run through intact peatlands delivered 350-470µg iron per liter creek water to seawater. To find out whether this humic-bound iron is bio-available to marine algae, we performed algal growth tests using the unicellular flagellated marine prymnesiophyte Diacronema lutheri and the unicellular marine green alga Chlorella salina, respectively. In both cases, the riverine humic material provided a highly bio-available source of iron to the marine algae. These results add a new item to the list of ecosystem services of Sphagnum-peatlands.