Feature-based molecular networking in the GNPS analysis environment.

Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.

Ultrahigh-Resolution Mass Spectrometry-Based Platform for Plasma Metabolomics Applied to Type 2 Diabetes Research.

Metabolomics-the endpoint of the omics cascade-is increasingly recognized as a preferred method for understanding the ultimate responses of biological systems to stress. Flow injection electrospray (FIE) mass spectrometry (MS) has advantages for untargeted metabolic fingerprinting due to its simplicity and capability for high-throughput screening but requires a high-resolution mass spectrometer to resolve metabolite features. In this study, we developed and validated a high-throughput and highly reproducible metabolomics platform integrating FIE with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS for analysis of both polar and nonpolar metabolite features from plasma samples. FIE-FTICR MS enables high-throughput detection of hundreds of metabolite features in a single mass spectrum without a front-end separation step. Using plasma samples from genetically identical obese mice with or without type 2 diabetes (T2D), we validated the intra and intersample reproducibility of our method and its robustness for simultaneously detecting alterations in both polar and nonpolar metabolite features. Only 5 min is needed to acquire an ultra-high resolution mass spectrum in either a positive or negative ionization mode. Approximately 1000 metabolic features were reproducibly detected and annotated in each mouse plasma group. For significantly altered and highly abundant metabolite features, targeted tandem MS (MS/MS) analyses can be applied to confirm their identity. With this integrated platform, we successfully detected over 300 statistically significant metabolic features in T2D mouse plasma as compared to controls and identified new T2D biomarker candidates. This FIE-FTICR MS-based method is of high throughput and highly reproducible with great promise for metabolomics studies toward a better understanding and diagnosis of human diseases.

Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.

INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample to be detected and quantified. Modern lipidomics techniques are ultra-sensitive assays that enable the discovery of novel biomarkers in a variety of fields and provide new insight in mechanistic investigations. Despite much progress in lipidomics, there remains, as for all high throughput "omics" strategies, the need to develop strategies to standardize and integrate quality control into studies in order to enhance robustness, reproducibility, and usability of studies within specific fields and beyond. OBJECTIVES: We aimed to understand how much results from lipid profiling in the model organism Caenorhabditis elegans are influenced by different culture conditions in different laboratories. METHODS: In this work we have undertaken an inter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans and daf-2(e1370) mutants lacking a functional insulin receptor. Sample were collected from worms grown in four separate laboratories under standardized growth conditions. We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MS analysis. RESULTS: We found common qualitative changes in several marker lipids in samples from the individual laboratories. On the other hand, even in this controlled experimental system, the exact fold-changes for each marker varied between laboratories. CONCLUSION: Our results thus reveal a serious limitation to the reproducibility of current lipid profiling experiments and reveal challenges to the integration of such data from different laboratories.

Using the knowns to discover the unknowns: MS-based dereplication uncovers structural diversity in 17-hydroxygeranyllinalool diterpene glycoside production in the Solanaceae.

Exploring the diversity of plant secondary metabolism requires efficient methods to obtain sufficient structural insights to discriminate previously known from unknown metabolites. De novo structure elucidation and confirmation of known metabolites (dereplication) remain a major bottleneck for mass spectrometry-based metabolomic workflows, and few systematic dereplication strategies have been developed for the analysis of entire compound classes across plant families, partly due to the complexity of plant metabolic profiles that complicates cross-species comparisons. 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) are abundant defensive secondary metabolites whose malonyl and glycosyl decorations are induced by jasmonate signaling in the ecological model plant Nicotiana attenuata. The multiple labile glycosidic bonds of HGL-DTGs result in extensive in-source fragmentation (IS-CID) during ionization. To reconstruct these IS-CID clusters from profiling data and identify precursor ions, we applied a deconvolution algorithm and created an MS/MS library from positive-ion spectra of purified HGL-DTGs. From this library, 251 non-redundant fragments were annotated, and a workflow to characterize leaf, flower and fruit extracts of 35 solanaceous species was established. These analyses predicted 105 novel HGL-DTGs that were restricted to Nicotiana, Capsicum and Lycium species. Interestingly, malonylation is a highly conserved step in HGL-DTG metabolism, but is differentially affected by jasmonate signaling among Nicotiana species. This MS-based workflow is readily applicable for cross-species re-identification/annotation of other compound classes with sufficient fragmentation knowledge, and therefore has the potential to support hypotheses regarding secondary metabolism diversification.

IncP-1ß plasmids of Comamonas sp. and Delftia sp. strains isolated from a wastewater treatment plant mediate resistance to and decolorization of the triphenylmethane dye crystal violet.

The application of toxic triphenylmethane dyes such as crystal violet (CV) in various industrial processes leads to large amounts of dye-contaminated sludges that need to be detoxified. Specific bacteria residing in wastewater treatment plants (WWTPs) are able to degrade triphenylmethane dyes. The objective of this work was to gain insights into the genetic background of bacterial strains capable of CV degradation. Three bacterial strains isolated from a municipal WWTP harboured IncP-1ß plasmids mediating resistance to and decolorization of CV. These isolates were assigned to the genera Comamonas and Delftia. The CV-resistance plasmid pKV29 from Delftia sp. KV29 was completely sequenced. In addition, nucleotide sequences of the accessory regions involved in conferring CV resistance were determined for plasmids pKV11 and pKV36 from the other two isolates. Plasmid pKV29 contains typical IncP-1ß backbone modules that are highly similar to those of previously sequenced IncP-1ß plasmids that confer antibiotic resistance, degradative capabilities or mercury resistance. The accessory regions located between the conjugative transfer (tra) and mating pair formation modules (trb) of all three plasmids analysed share common modules and include a triphenylmethane reductase gene, tmr, that is responsible for decolorization of CV. Moreover, these accessory regions encode other enzymes that are dispensable for CV degradation and hence are involved in so-far-unknown metabolic pathways. Analysis of plasmid-mediated degradation of CV in Escherichia coli by ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight MS revealed that leuco crystal violet was the first degradation product. Michler's ketone and 4-dimethylaminobenzaldehyde appeared as secondary degradation metabolites. Enzymes encoded in the E. coli chromosome seem to be responsible for cleavage of leuco crystal violet. Plasmid-mediated degradation of triphenylmethane dyes such as CV is an option for the biotechnological treatment of sludges contaminated with these dyes.

Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

Olive Oil Quality and Authenticity Assessment Aspects Employing FIA-MRMS and LC-Orbitrap MS Metabolomic Approaches.

Edible vegetable oils comprise integral components of humans' daily diet during the lifetime. Therefore, they constitute a central part of dietary-exposome, which among other factors regulates human health. In particular, the regular consumption of olive oil (OO) has been largely accepted as a healthy dietary pattern. Responsible for its recognition as a superior edible oil is its exceptional aroma and flavor. Its unique composition is characterized by high levels of monounsaturated fatty acids and the presence of minor constituents with important biological properties, such as the so-called OO polyphenols. Being a high added value product, OO suffers from extensive fraud and adulteration phenomena. However, its great chemical complexity, variability, and the plethora of parameters affecting OO composition hamper significantly the selection of the absolute criteria defining quality and authenticity, and a reliable and robust methodology is still unavailable. In the current study, Flow Injection Analysis-Magnetic Resonance Mass Spectrometry (FIA-MRMS) was investigated under a metabolic profiling concept for the analysis of Greek Extra Virgin Olive Oils (EVOO). More than 200 monovarietal (Koroneiki) EVOO samples were collected from the main Greek OO producing regions and investigated. Both intact oil and the corresponding polyphenols were analyzed in fast analysis time of 2 and 8 min, respectively. In parallel, an LC-Orbitrap MS platform was used to verify the efficiency of the method as well as a tool to increase the identification confidence of the proposed markers. Based on the results, with FIA-MRMS, comparable and improved projection and prediction models were generated in comparison to those of the more established LC-MS methodology. With FIA-MRMS more statistically significant compounds and chemical classes were identified as quality and authenticity markers, associated with specific parameters, i.e. geographical region, cultivation practice, and production procedure. Furthermore, it was possible to monitor both lipophilic and hydrophilic compounds with a single analysis. To our knowledge, this approach is among the few studies in which two FT-MS platforms combining LC and FIA methods were integrated to provide solutions to quality control aspects of OO. Moreover, both lipophilic and hydrophilic components are analyzed together, providing a holistic quality control workflow for OO.

Trapped ion mobility spectrometry and PASEF enable in-depth lipidomics from minimal sample amounts.

A comprehensive characterization of the lipidome from limited starting material remains very challenging. Here we report a high-sensitivity lipidomics workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry (TIMS). Taking advantage of parallel accumulation-serial fragmentation (PASEF), we fragment on average 15 precursors in each of 100 ms TIMS scans, while maintaining the full mobility resolution of co-eluting isomers. The acquisition speed of over 100 Hz allows us to obtain MS/MS spectra of the vast majority of isotope patterns. Analyzing 1 µL of human plasma, PASEF increases the number of identified lipids more than three times over standard TIMS-MS/MS, achieving attomole sensitivity. Building on high intra- and inter-laboratory precision and accuracy of TIMS collisional cross sections (CCS), we compile 1856 lipid CCS values from plasma, liver and cancer cells. Our study establishes PASEF in lipid analysis and paves the way for sensitive, ion mobility-enhanced lipidomics in four dimensions.

Generation of a Collision Cross Section Library for Multi-Dimensional Plant Metabolomics Using UHPLC-Trapped Ion Mobility-MS/MS.

The utility of metabolomics is well documented; however, its full scientific promise has not yet been realized due to multiple technical challenges. These grand challenges include accurate chemical identification of all observable metabolites and the limiting depth-of-coverage of current metabolomics methods. Here, we report a combinatorial solution to aid in both grand challenges using UHPLC-trapped ion mobility spectrometry coupled to tandem mass spectrometry (UHPLC-TIMS-TOF-MS). TIMS offers additional depth-of-coverage through increased peak capacities realized with the multi-dimensional UHPLC-TIMS separations. Metabolite identification confidence is simultaneously enhanced by incorporating orthogonal collision cross section (CCS) data matching. To facilitate metabolite identifications, we created a CCS library of 146 plant natural products. This library was generated using TIMS with N2 drift gas to record the TIMSCCSN2 of plant natural products with a high degree of reproducibility; i.e., average RSD = 0.10%. The robustness of TIMSCCSN2 data matching was tested using authentic standards spiked into complex plant extracts, and the precision of CCS measurements were determined to be independent of matrix affects. The utility of the UHPLC-TIMS-TOF-MS/MS in metabolomics was then demonstrated using extracts from the model legume Medicago truncatula and metabolites were confidently identified based on retention time, accurate mass, molecular formula, and CCS.

Investigation of central carbon metabolism and the 2-methylcitrate cycle in Corynebacterium glutamicum by metabolic profiling using gas chromatography-mass spectrometry.

The 2-methylcitrate cycle as the primary way to metabolize propionate was investigated using metabolic profiling. For this purpose, a fast harvesting procedure was applied in which cells growing in liquid minimal medium were harvested by a short centrifugation and freeze-dried. Subsequently, gas chromatography-mass spectrometry of polar extracts derivatized by MSTFA was employed for metabolite characterization. Routinely more than 300 different peaks were obtained in the chromatograms, and 74 substances were identified unequivocally by using pure standards. The procedure provided reliable data which closely relate to prior knowledge on flux distributions during growth on glucose and acetate as carbon sources. Propionate degradation via the 2-methylcitrate cycle was demonstrated on the metabolite level by the detection of the intermediates 2-methylcitrate and 2-methylisocitrate. Further characterization of the 2-methylcitrate cycle was carried out by comparing different mutant strains of this pathway. The growth deficit of a prpD2-mutant strain observed when propionate is added to a culture growing on acetate indicates that the toxic effect of propionate is based on the accumulation of 2-methylcitrate. It could also be shown that the 2-methylcitrate cycle is active in the absence of propionate and might fulfill house-keeping functions in the degradation of fatty acids or branched-chain amino acids.

The Plant Immunity Regulating F-Box Protein CPR1 Supports Plastid Function in Absence of Pathogens.

The redox imbalanced 6 mutant (rimb6) of Arabidopsis thaliana was isolated in a genetic screening approach for mutants with defects in chloroplast-to-nucleus redox signaling. It has an atypically low activation status of the 2-Cys peroxiredoxin-A promoter in the seedling stage. rimb6 shows wildtype-like germination, seedling development and greening, but slower growth and reduced biomass in the rosette stage. Mapping of the casual mutation revealed that rimb6 carries a single nucleotide polymorphism in the gene encoding CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED (PR) GENES 1, CPR1 (At4g12560), leading to a premature stop codon. CPR1 is known as a repressor of pathogen signaling and regulator of microtubule organization. Allelism of rimb6 and cpr1 revealed a function of CPR1 in chloroplast stress protection. Expression studies in pathogen signaling mutants demonstrated that CPR1-mediated activation of genes for photosynthesis and chloroplast antioxidant protection is, in contrast to activation of pathogen responses, regulated independently from PAD4-controlled salicylic acid (SA) accumulation. We conclude that the support of plastid function is a basic, SA-independent function of CPR1.

Metabolite profiles of nodulated alfalfa plants indicate that distinct stages of nodule organogenesis are accompanied by global physiological adaptations.

An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago sativa is dependent on a balanced physiological interaction enabling the microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic interaction is regulated by still poorly understood control mechanisms. A first step toward a better understanding of nodule metabolism was the determination of characteristic metabolites for alfalfa root nodules. Furthermore, nodules arrested at different developmental stages were analyzed in order to address metabolic changes induced during the progression of nodule formation. Metabolite profiles of bacteroid-free pseudonodule extracts indicated that early nodule developmental processes are accompanied by photosynthate translocation but no massive organic acid formation. To determine metabolic adaptations induced by the presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains lacking the nitrogenase protein were analyzed. The bacteroids are unable to provide ammonium to the host plant, which is metabolically reflected by reduced levels of characteristic amino acids involved in ammonium fixation. Elevated levels of starch and sugars in Fix(-) nodules provide strong evidence that plant sanctions preventing a transformation from a symbiotic to a potentially parasitic interaction are not strictly realized via photosynthate supply. Instead, metabolic and gene expression data indicate that alfalfa plants react to nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis and an early induction of senescence. Noneffective symbiotic interactions resulting from plants nodulated by mutant rhizobia also are reflected in characteristic metabolic changes in leaves. These are typical for nitrogen deficiency, but also highlight metabolites potentially involved in sensing the N status.

GC-MS based metabolite profiling implies three interdependent ways of ammonium assimilation in Medicago truncatula root nodules.

In symbiotic interaction with legume plants, bacteria termed Rhizobia can fix massive amounts of atmospheric nitrogen which is primarily provided in the form of ammonium to the host plants. Therefore, legume root nodules that house the symbiotic bacteria are ideally suited to study the process of primary ammonium assimilation. Here, we present a GC-MS based metabolite profiling analysis of Medicago truncatula root nodules (induced by the bacterium Sinorhizobium meliloti) before and after inhibition of glutamine synthetase (GS) by the chemical herbicide phosphinotricine. The primary role of GS in ammonium assimilation was revealed by drastically reduced levels of glutamine in phosphinotricine treated root nodules. In comparison to previous results of increased asparagine synthetase transcript and protein abundances in GS inhibited nodules the metabolic data revealed that decreased amounts of aspartate might preclude taking advantage of this elevated enzymatic activity. A potential role of glutamate dehydrogenase in ammonium assimilation was metabolically indicated 24 and 48 h after GS inhibition. Therefore, nodule ammonium assimilation might in principle involve three interdependent metabolic pathways which are adjusted to control basic nitrogen metabolism.

Metabolite profiling of wheat grains (Triticum aestivum L.) from organic and conventional agriculture.

In some European community countries up to 8% of the agricultural area is managed organically. The aim was to obtain a metabolite profile for wheat (Triticum aestivum L.) grains grown under comparable organic and conventional conditions. These conditions cannot be found in plant material originating from different farms or from products purchased in supermarkets. Wheat grains from a long-term biodynamic, bioorganic, and conventional farming system from the harvest 2003 from Switzerland were analyzed. The presented data show that using a high throughput GC-MS technique, it was possible to determine relative levels of a set of 52 different metabolites including amino acids, organic acids, sugars, sugar alcohols, sugar phosphates, and nucleotides from wheat grains. Within the metabolites from all field trials, there was at the most a 50% reduction comparing highest and lowest mean values. The statistical analysis of the data shows that the metabolite status of the wheat grain from organic and mineralic farming did not differ in concentrations of 44 metabolites. This result indicates no impact or a small impact of the different farming systems. In consequence, we did not detect extreme differences in metabolite composition and quality of wheat grains.

Physiological and molecular characterization of a Synechocystis sp. PCC 6803 mutant lacking histidine kinase Slr1759 and response regulator Slr1760.

The hybrid sensory histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. strain PCC 6803 contains multiple sensory domains and a multi-step phosphorelay system. Immuno blot analysis provided evidence that the histidine kinase Slr1759 is associated with the cytoplasmic membrane. The gene slr1759 is part of an operon together with slr1760, encoding a response regulator. A comparative investigation was performed on Synechocystis sp. strain PCC 6803 wild type (WT) and an insertionally inactivated slr1759-mutant (Hik14) which also lacks the transcript for the response regulator Slr1760. The mutant Hik14 grew significantly slower than WT in the early growth phase, when both were inoculated with a low cell density into BG11 medium without additional buffer and when aerated with air enriched with 2% CO2. Since the aeration with CO2-enriched air results in a decrease of the pH value in the medium, the growth experiments indicated that Hik14 is not able to adjust its metabolic activities as rapidly as WT to compensate for a larger decrease of the pH value in the medium. No significant differences in growth between Hikl4 and WT were observed when cells were inoculated with a higher cell density in BG11 medium or when the BG11 medium contained 50 mM Epps-NaOH, pH 7.5, to prevent the pH drop. This Hik14 phenotype has so far only been seen under the above defined growth condition. Results of photosynthetic activity measurements as well as Northern blot-, immuno blot-, and metabolite analyses suggest that the two-component system Slr1759/Slr1760 has a function in the coordination of several metabolic activities which is in good agreement with the complex domain structure of Slr1759. The direct targets of this two-component system have so far not been identified.

Untargeted mass spectrometric approach in metabolic healthy offspring of patients with type 2 diabetes reveals medium-chain acylcarnitine as potential biomarker for lipid induced glucose intolerance (LGIT).

Offspring of type 2 diabetes (T2D) patients have increased risk to develop diabetes, due to inherited genetic susceptibility that directly interferes with the individual adaption to environmental conditions. We characterise T2D offspring (OSP) to identify metabolic risk markers for early disease prediction. Plasma of metabolically healthy OSP individuals (n = 43) was investigated after an oral lipid tolerance test (oLTT) by an untargeted mass spectrometric approach for holistic metabolome analyses. Two subgroups of OSP probands can be separated by oLTT, although not differing in general clinical parameters. Analyses of the plasma metabolome revealed mainly medium-chain acylcarnitines and very long-chain fatty acids with differential abundance in the subgroups. The study presented indicates that metabolically healthy OSP of T2D patients differ upon metabolic challenging in serum metabolite composition, especially medium-chain acylcarnitines. The difference suggest that postprandial lipid induced glucose intolerance (LGIT) may serve as a further valuable marker for early diabetes prediction.

Comprehensive metabolite profiling of Sinorhizobium meliloti using gas chromatography-mass spectrometry.

A metabolite analysis of the soil bacterium Sinorhizobium meliloti was established as a first step towards a better understanding of the symbiosis with its host plant Medicago truncatula. A crucial step was the development of fast harvesting and extraction methods for the bacterial metabolites because of rapid changes in their composition. S. meliloti 1021 cell cultures grown in minimal medium were harvested by centrifugation, filtration or immediate freezing in liquid nitrogen followed by a lyophilisation step. Bacteria were lysed mechanically in methanol and hydrophilic compounds were analysed after methoxymation and silylisation via GC-MS. The different compounds were identified by comparison with the NIST 98 database and available standards. From about 200 peaks in each chromatogram 65 compounds have been identified so far. A comparison of the different extraction methods giving the metabolite composition revealed clear changes in several amino acids and amino acid precursor pools. A principal component analysis (PCA) was able to distinguish S. meliloti cells grown on different carbon sources based on their metabolite profile. A comparison of the metabolite composition of a S. meliloti leucine auxotrophic mutant with the wild type revealed a marked accumulation of 2-isopropylmalate in the mutant. Interestingly, the accumulated metabolite is not the direct substrate of the mutated enzyme, 3-isopropylmalate dehydrogenase, but the substrate of isopropylmalate isomerase, which acts one step further upstream in the biosynthetic pathway of leucine. This finding further emphasises the importance of integrating metabolic data into post-genomic research.

A novel ion pairing LC/MS metabolomics protocol for study of a variety of biologically relevant polar metabolites.

We report a method of ion-pairing liquid chromatography coupled to mass spectrometry (IP-LC-MS) that we have developed for the sensitive detection and quantification of a variety of biologically relevant polar molecules. We use the ion-pairing agent diamyl ammonium to improve chromatographic resolution of polar compounds, such as nucleotide cofactors, sugar phosphates, and organic acids, that are generally poorly retained by conventional reverse phase chromatographic methods. This method showed good linearity (average R value of 0.996) and reproducibility (generally RSD values <10%). We demonstrate the utility of this method by investigating the metabolomic signature of three distinct biological systems: the metabolic response to lack of superoxide dismutase activity and to paraquat induced oxidative stress, and the metabolic profiles of four different Drosophila species.