Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.

Unravelling the seasonal dynamics of the metabolome of white asparagus spears using untargeted metabolomics.

INTRODUCTION: The white asparagus season lasts 4 months while the harvest period per field is 8 weeks. Different varieties are better suited for harvesting early or late in the season. Little is known of the dynamics of secondary metabolites of white asparagus during the production season. OBJECTIVE: Characterization of the metabolome of white asparagus spears covering volatile and non-volatile composition in relation to quality aspects. METHODS: Eight varieties, harvested repeatedly during two consecutive seasons were analysed following an untargeted metabolomics workflow using SPME GC-MS and LC-MS. Linear regression, cluster and network analyses were used to explore the profile dynamics, unravel patterns and study the influence of genotype and environment. RESULTS: The metabolite profiles were influenced by the harvest moment and genetic background. Metabolites that significantly changed over time were distributed across seven clusters based on their temporal patterns. Two clusters including monoterpenes, benzenoids and saponins showed the most prominent seasonal changes. The changes depicted by the other five clusters were mainly ≤ 2-fold relative to the harvest start. Known asparagus aroma compounds were found to be relatively stable across the season/varieties. Heat-enhanced cultivation appeared to yield spears early in season with a similar metabolome to those harvested later. CONCLUSION: The dynamics of the white asparagus metabolome is influenced by a complex relationship between the onset of spear development, the moment of harvest and the genetic background. The typical perceived asparagus flavour profile is unlikely to be significantly affected by these dynamics.

How Individuals Use Metaphors to Negotiate Fertility Treatment Decision-Making with Their Romantic Partners.

Fertility problems, or the inability to conceive or carry a pregnancy to term for a period of over 12 months while engaging in unprotected sex, affects 12% of women and 9% of men of childbearing age. To answer calls for more research about individuals' fertility decision-making (DM) with their partners, we conducted in-depth, semi-structured interviews with 53 individuals who have experienced fertility decision-making with a romantic partner at some point in their lives. Our findings indicate at least three primary ways individuals and their partners navigated their decision-making communication in their infertility "journeys:" (1) the Driver-Navigator, (2) Driver-Passenger, and (3) Driver-Backseat Driver approaches. All decision-making communication approaches were viewed by individuals as collaborative (i.e. shared), but varied in degrees of "togetherness" (high, moderate, low) in how they communicated with each other about treatment decisions. Implications include helping couples and their clinicians to be aware of their DM approach(es) and offering alternative DM approaches based on understanding how and why certain approaches may (not) be effective in addressing goals, needs, and identities.

Metabolomics should be deployed in the identification and characterization of gene-edited crops.

Gene-editing techniques are currently revolutionizing biology, allowing far greater precision than previous mutagenic and transgenic approaches. They are becoming applicable to a wide range of plant species and biological processes. Gene editing can rapidly improve a range of crop traits, including disease resistance, abiotic stress tolerance, yield, nutritional quality and additional consumer traits. Unlike transgenic approaches, however, it is not facile to forensically detect gene-editing events at the molecular level, as no foreign DNA exists in the elite line. These limitations in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process in itself. Rapid advances in sequencing and genome assembly increasingly facilitate genome sequencing as a means of characterizing new varieties generated by gene-editing techniques. Nevertheless, subtle edits such as single base changes or small deletions may be difficult to distinguish from normal variation within a genotype. Given these emerging scenarios, downstream 'omics' technologies reflective of edited affects, such as metabolomics, need to be used in a more prominent manner to fully assess compositional changes in novel foodstuffs. To achieve this goal, metabolomics or 'non-targeted metabolite analysis' needs to make significant advances to deliver greater representation across the metabolome. With the emergence of new edited crop varieties, we advocate: (i) concerted efforts in the advancement of 'omics' technologies, such as metabolomics, and (ii) an effort to redress the use of the technology in the regulatory assessment for metabolically engineered biotech crops.

Systematic selection of competing metabolomics methods in a metabolite-sensory relationship study.

INTRODUCTION: The relationship between the chemical composition of food products and their sensory profile is a complex association confronting many challenges. However, new untargeted methodologies are helping correlate metabolites with sensory characteristics in a simpler manner. Nevertheless, in the pilot phase of a project, where only a small set of products are used to explore the relationships, choices have to be made about the most appropriate untargeted metabolomics methodology. OBJECTIVE: To provide a framework for selecting a metabolite-sensory methodology based on: the quality of measurements, the relevance of the detected metabolites in terms of distinguishing between products or in terms of whether they can be related to the sensory attributes of the products. METHODS: In this paper we introduce a systematic approach to explore all these different aspects driving the choice for the most appropriate metabolomics method. RESULTS: As an example we have used a tomato soup project where the choice between two sampling methods (SPME and SBSE) had to be made. The results are not always consistently pointing to the same method as being the best. SPME was able to detect metabolites with a better precision, SBSE seemed to be able to provide a better distinction between the soups. CONCLUSION: The three levels of comparison provide information on how the methods could perform in a follow up study and will help the researcher to make a final selection for the most appropriate method based on their strengths and weaknesses.

Amicus cum Laude: Ethnodramatic Inquiry Regarding Sharing Mental Health in College.

In this manuscript, I utilize an ethnodramatic methodology in reanalyzing two data sets about college friends disclosing and receiving mental health-related information. After describing ethnodrama and how this methodology applies to mental health-related inquiry, I detail my process of creating an ethnodrama from two extant data sets. The result is an ethnodrama called Amicus cum Laude: Becoming a Friend with Honor for Mental Illness, a one-act play about how friends discuss mental health issues with one another. After providing the ethnodrama, I offer recommendations for taking the ethnodrama from page to stage while reflecting on and critiquing the final product.

Transcription Factor-Mediated Control of Anthocyanin Biosynthesis in Vegetative Tissues.

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology.

Mass spectrometry-based metabolomics of volatiles as a new tool for understanding aroma and flavour chemistry in processed food products.

BACKGROUND: When foods are processed or cooked, many chemical reactions occur involving a wide range of metabolites including sugars, amino acids and lipids. These chemical processes often lead to the formation of volatile aroma compounds that can make food tastier or may introduce off-flavours. Metabolomics tools are only now being used to study the formation of these flavour compounds in order to understand better the beneficial and less beneficial aspects of food processing. AIM OF REVIEW: To provide a critical overview of the diverse MS-based studies carried out in recent years in food metabolomics and to review some biochemical properties and flavour characteristics of the different groups of aroma-related metabolites. A description of volatiles from processed foods, and their relevant chemical and sensorial characteristics is provided. In addition, this review also summarizes the formation of the flavour compounds from their precursors, and the interconnections between Maillard reactions and the amino acid, lipid, and carbohydrate degradation pathways. KEY SCIENTIFIC CONCEPTS OF REVIEW: This review provides new insights into processed ingredients and describes how metabolomics will help to enable us to produce, preserve, design and distribute higher-quality foods for health promotion and better flavour.

The Effect of Calcium Buffering and Calcium Sensor Type on the Sensitivity of an Array-Based Bitter Receptor Screening Assay.

The genetically encoded calcium sensor protein Cameleon YC3.6 has previously been applied for functional G protein-coupled receptor screening using receptor cell arrays. However, different types of sensors are available, with a wide range in [Ca2+] sensitivity, Hill coefficients, calcium binding domains, and fluorophores, which could potentially improve the performance of the assay. Here, we compared the responses of 3 structurally different calcium sensor proteins (Cameleon YC3.6, Nano140, and Twitch2B) simultaneously, on a single chip, at different cytosolic expression levels and in combination with 2 different bitter receptors, TAS2R8 and TAS2R14. Sensor concentrations were modified by varying the amount of calcium sensor DNA that was printed on the DNA arrays prior to reverse transfection. We found that ~2-fold lower concentrations of calcium sensor protein, by transfecting 4 times less sensor-coding DNA, resulted in more sensitive bitter responses. The best results were obtained with Twitch2B, where, relative to YC3.6 at the default DNA concentration, a 4-fold lower DNA concentration increased sensitivity 60-fold and signal strength 5- to 10-fold. Next, we compared the performance of YC3.6 and Twitch2B against an array with 11 different bitter taste receptors. We observed a 2- to 8-fold increase in sensitivity using Twitch2B compared with YC3.6. The bitter receptor arrays contained 300 spots and could be exposed to a series of 18 injections within 1 h resulting in 5400 measurements. These optimized sensor conditions provide a basis for enhancing receptomics calcium assays for receptors with poor Ca2+ signaling and will benefit future high-throughput receptomics experiments.

Engineering de novo anthocyanin production in Saccharomyces cerevisiae.

BACKGROUND: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. RESULTS: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively. CONCLUSION: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System.

Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.

Processing black mulberry into jam: effects on antioxidant potential and in vitro bioaccessibility.

BACKGROUND: Black mulberries (Morus nigra) were processed into jam on an industrialised scale, including the major steps of: selection of frozen black mulberries, adding glucose-fructose syrup and water, cooking, adding citric acid and apple pectin, removing seeds, and pasteurisation. Qualitative and quantitative determinations of antioxidants in black mulberry samples were performed using spectrophotometric methods, as well as HPLC- and LC-QTOF-MS-based measurements. These analyses included the determination of total polyphenolic content, % polymeric colour, total and individual anthocyanin contents, antioxidant capacity, and in vitro bioaccessibility in processing samples. RESULTS: Jam processing led to a significant reduction in total phenolics (88%), total flavonoids (89%), anthocyanins (97%), and antioxidant capacity (88-93%) (P < 0.05). Individual anthocyanin contents, determined using HPLC analysis, also showed a significant decrease (∼99% loss). In contrast, % recovery of bioaccessible total phenolics, anthocyanins, and antioxidant capacity (ABTS assay) increased after jam processing (16%, 12%, and 37%, respectively). CONCLUSION: Fruit processing resulted in losses of polyphenols, anthocyanins, and antioxidant capacity of black mulberry jam. Optimisation of food processing could help to protect the phenolic compounds in fruits which might be helpful for the food industry to minimise the antioxidant loss and improve the final product quality. © 2016 Society of Chemical Industry.

Spatially Resolved Plant Metabolomics: Some Potentials and Limitations of Laser-Ablation Electrospray Ionization Mass Spectrometry Metabolite Imaging.

Laser-ablation electrospray ionization (LAESI)-mass spectrometry imaging has been applied to contrasting plant organs to assess its potential as a procedure for performing in vivo metabolomics in plants. In a proof-of-concept experiment, purple/white segmented Phalaenopsis spp. petals were first analyzed using standard liquid chromatography-mass spectrometry analyses of separate extracts made specifically from the purple and white regions. Discriminatory compounds were defined and putatively annotated. LAESI analyses were then performed on living tissues, and these metabolites were then relocalized within the LAESI-generated data sets of similar tissues. Maps were made to illustrate their locations across the petals. Results revealed that, as expected, anthocyanins always mapped to the purple regions. Certain other (nonvisible) polyphenols were observed to colocalize with the anthocyanins, whereas others were found specifically within the white tissues. In a contrasting example, control and Cladosporium fulvum-infected tomato (Solanum lycopersicum) leaves were subjected to the same procedures, and it could be observed that the alkaloid tomatine has clear heterogeneous distribution across the tomato leaf lamina. Furthermore, LAESI analyses revealed perturbations in alkaloid content following pathogen infection. These results show the clear potential of LAESI-based imaging approaches as a convenient and rapid way to perform metabolomics analyses on living tissues. However, a range of limitations and factors have also been identified that must be taken into consideration when interpreting LAESI-derived data. Such aspects deserve further evaluation before this approach can be applied in a routine manner.

Non-smoky glycosyltransferase1 prevents the release of smoky aroma from tomato fruit.

Phenylpropanoid volatiles are responsible for the key tomato fruit (Solanum lycopersicum) aroma attribute termed "smoky." Release of these volatiles from their glycosylated precursors, rather than their biosynthesis, is the major determinant of smoky aroma in cultivated tomato. using a combinatorial omics approach, we identified the non-smoky glycosyltransferase1 (NSGT1) gene. Expression of NSGT1 is induced during fruit ripening, and the encoded enzyme converts the cleavable diglycosides of the smoky-related phenylpropanoid volatiles into noncleavable triglycosides, thereby preventing their deglycosylation and release from tomato fruit upon tissue disruption. In an nsgt1/nsgt1 background, further glycosylation of phenylpropanoid volatile diglycosides does not occur, thereby enabling their cleavage and the release of corresponding volatiles. Using reverse genetics approaches, the NSGT1-mediated glycosylation was shown to be the molecular mechanism underlying the major quantitative trait locus for smoky aroma. Sensory trials with transgenic fruits, in which the inactive nsgt1 was complemented with the functional NSGT1, showed a significant and perceivable reduction in smoky aroma. NSGT1 may be used in a precision breeding strategy toward development of tomato fruits with distinct flavor phenotypes.

A Review on the Effect of Drying on Antioxidant Potential of Fruits and Vegetables.

The role of antioxidants in human nutrition has gained increased interest, especially due to their associated health beneficial effects for a number of chronic diseases, including cardiovascular diseases and certain types of cancer. Fruits and vegetables are perishable and difficult to preserve as fresh products. Dried fruits and vegetables can be easily stored, transported at relatively low cost, have reduced packing costs, and their low water content delays microbial spoilage. Air-, freeze-, microwave- and sun-drying are among the most thoroughly studied drying methods. This review provides an overview of recent findings on the effects of different drying techniques on major antioxidants of fruits and vegetables. In particular, changes in ascorbic acid, carotenoids, flavonoids, phenolic acids, total phenolics, and antioxidant activity are discussed in detail.

An O-methyltransferase modifies accumulation of methylated anthocyanins in seedlings of tomato.

Anthocyanins contribute to the appearance of fruit by conferring to them a red, blue or purple colour. In a food context, they have also been suggested to promote consumer health. In purple tomato tissues, such as hypocotyls, stems and purple fruits, various anthocyanins accumulate. These molecules have characteristic patterns of modification, including hydroxylations, methylations, glycosylations and acylations. The genetic basis for many of these modifications has not been fully elucidated, and nor has their role in the functioning of anthocyanins. In this paper, AnthOMT, an O-methyltransferase (OMT) mediating the methylation of anthocyanins, has been identified and functionally characterized using a combined metabolomics and transcriptomics approach. Gene candidates were selected from the draft tomato genome, and their expression was subsequently monitored in a tomato seedling system comprising three tissues and involving several time points. In addition, we also followed gene expression in wild-type red and purple transgenic tomato fruits expressing Rosea1 and Delila transcription factors. Of the 57 candidates identified, only a single OMT gene showed patterns strongly correlating with both accumulation of anthocyanins and expression of anthocyanin biosynthesis genes. This candidate (AnthOMT) was compared to a closely related caffeoyl CoA OMT by recombinant expression in Escherichia coli, and then tested for substrate specificity. AnthOMT showed a strong affinity for glycosylated anthocyanins, while other flavonoid glycosides and aglycones were much less preferred. Gene silencing experiments with AnthOMT resulted in reduced levels of the predominant methylated anthocyanins. This confirms the role of this enzyme in the diversification of tomato anthocyanins.

The genetics of plant metabolism.

Variation for metabolite composition and content is often observed in plants. However, it is poorly understood to what extent this variation has a genetic basis. Here, we describe the genetic analysis of natural variation in the metabolite composition in Arabidopsis thaliana. Instead of focusing on specific metabolites, we have applied empirical untargeted metabolomics using liquid chromatography-time of flight mass spectrometry (LC-QTOF MS). This uncovered many qualitative and quantitative differences in metabolite accumulation between A. thaliana accessions. Only 13.4% of the mass peaks were detected in all 14 accessions analyzed. Quantitative trait locus (QTL) analysis of more than 2,000 mass peaks, detected in a recombinant inbred line (RIL) population derived from the two most divergent accessions, enabled the identification of QTLs for about 75% of the mass signals. More than one-third of the signals were not detected in either parent, indicating the large potential for modification of metabolic composition through classical breeding.

δ13C as a tool for iron and phosphorus deficiency prediction in crops.

Many studies proposed the use of stable carbon isotope ratio (δ13C) as a predictor of abiotic stresses in plants, considering only drought and nitrogen deficiency without further investigating the impact of other nutrient deficiencies, that is, phosphorus (P) and/or iron (Fe) deficiencies. To fill this knowledge gap, we assessed the δ13C of barley (Hordeum vulgare L.), cucumber (Cucumis sativus L.), maize (Zea mays L.), and tomato (Solanum lycopersicon L.) plants suffering from P, Fe, and combined P/Fe deficiencies during a two-week period using an isotope-ratio mass spectrometer. Simultaneously, plant physiological status was monitored with an infra-red gas analyzer. Results show clear contrasting time-, treatment-, species-, and tissue-specific variations. Furthermore, physiological parameters showed limited correlation with δ13C shifts, highlighting that the plants' δ13C, does not depend solely on photosynthetic carbon isotope fractionation/discrimination (Δ). Hence, the use of δ13C as a predictor is highly discouraged due to its inability to detect and discern different nutrient stresses, especially when combined stresses are present.

Effect of drying treatments on the global metabolome and health-related compounds in tomatoes.

Drying fruits and vegetables is a long-established preservation method, and for tomatoes, in most cases sun-drying is preferred. Semi-drying is relatively a new application aimed to preserve better the original tomato properties. We have assessed the effects of different drying methods on the phytochemical variation in tomato products using untargeted metabolomics and targeted analyses of key compounds. An LC-MS approach enabled the relative quantification of 890 mostly semi-polar secondary metabolites and GC-MS analysis in the relative quantification of 270 polar, mostly primary metabolites. Metabolite profiles of sun-dried and oven-dried samples were clearly distinct and temperature-dependent. Both treatments caused drastic changes in lycopene and vitamins with losses up to > 99% compared to freeze-dried controls. Semi-drying had less impact on these compounds. In vitro bioaccessibility analyses of total phenolic compounds and antioxidants in a gastrointestinal digestion protocol revealed the highest recovery rates in semi-dried fruits. Semi-drying is a better way of preserving tomato phytochemicals, based on both composition and bioaccessibility results.

Metabolomics and sensory evaluation of white asparagus ingredients in instant soups unveil important (off-)flavours.

Split-stream processing of asparagus waste stream is a novel approach to produce spray-dried powder and fibre. Asparagus ingredients processed by this method and a commercial asparagus powder were compared by evaluating their flavour profile in a soup formulation. Professional sensory panel and untargeted metabolomics approaches using GC-MS and LC-MS were carried out. Unsupervised and supervised statistical analyses were performed to highlight discriminatory metabolites and correlate these to sensory attributes. The spray-dried powder scored higher on asparagus flavour compared to the commercial powder. The fibre negatively impacted the taste and mouthfeel of the soups. GC-O-MS confirmed the role of dimethyl sulphide, 2-methoxy-3-isopropyl pyrazine and 2-methoxy-3-isobutyl pyrazine in asparagus odour. Seven new volatile compounds are also proposed to contribute to asparagus flavour notes, most of which were more abundant in the spray-dried powder. This research demonstrates the feasibility of upcycling asparagus waste streams into flavour-rich ingredients with good sensorial properties.