Wageningen Research zoekt nieuwe toekomst
Bino, Raoul ; Smaal, Aad ; Bakker, Sjaak ; Daane, Arjen - \ 2016
Het Wageningse contractonderzoek is in crisis. Ouwe trouwe opdrachtgevers zijn weggevallen, nieuwe verdienmodellen zijn nodig. Een werkgroep onder leiding van Raoul Bino denkt hier nu over na. De eerste succesvolle voorbeelden zijn er.
|Als twee instituten van naam en faam de handen ineenslaan : gezamenlijke innovaties op het gebied van gezonde voeding en biobased
Binnendijk, D. ; Bino, R.J. ; Zondervan, C. - \ 2015
TNOTIME 3 (2015)najaar. - p. 6 - 9.
Food & biobased research : Healthy and sustainable choices : now and in the future
Bino, R.J. ; Gorselink, M. ; Seventer, E. van; Yilmaz, G. ; Timmermans, A.J.M. ; Amerongen, A. van; Bos, H.L. ; Haveren, J. van; Westra, E.H. ; Wijk, R.A. de; Bolck, C.H. ; Boerrigter, H.A.M. ; Matser, A.M. ; Barbosa, M.J. ; Peppelenbos, H.W. ; Maat, H.W. ter; Klemm, W. - \ 2014
Wageningen UR FBR
onderzoeksinstituten - onderzoek - voedingsonderzoek - biobased economy - ketenmanagement - bioraffinage - chemie op basis van biologische grondstoffen - materialen uit biologische grondstoffen - consumentenwetenschappen - research institutes - research - nutrition research - biobased economy - supply chain management - biorefinery - biobased chemistry - biobased materials - consumer sciences
Corporate brochure of Food & Biobased Research (FBR) Wageningen UR.
Assessment of pleiotropic transcriptome perturbations in Arabidopsis engineered for indirect insect defence
Houshyani Hassanzadeh, B. ; Krol, A.R. van der; Bino, R.J. ; Bouwmeester, H.J. - \ 2014
BMC Plant Biology 14 (2014). - ISSN 1471-2229
global gene-expression - metabolomics - thaliana - gm - biosynthesis - metabolism - emission - synthase - crops - wheat
Background: Molecular characterization is an essential step of risk/safety assessment of genetically modified (GM) crops. Holistic approaches for molecular characterization using omics platforms can be used to confirm the intended impact of the genetic engineering, but can also reveal the unintended changes at the omics level as a first assessment of potential risks. The potential of omics platforms for risk assessment of GM crops has rarely been used for this purpose because of the lack of a consensus reference and statistical methods to judge the significance or importance of the pleiotropic changes in GM plants. Here we propose a meta data analysis approach to the analysis of GM plants, by measuring the transcriptome distance to untransformed wild-types. Results: In the statistical analysis of the transcriptome distance between GM and wild-type plants, values are compared with naturally occurring transcriptome distances in non-GM counterparts obtained from a database. Using this approach we show that the pleiotropic effect of genes involved in indirect insect defence traits is substantially equivalent to the variation in gene expression occurring naturally in Arabidopsis. Conclusion: Transcriptome distance is a useful screening method to obtain insight in the pleiotropic effects of genetic modification.
In Silico Prediction and Automatic LC–MSn Annotation of Green Tea Metabolites in Urine
Ridder, L.O. ; Hooft, J.J.J. van der; Verhoeven, S. ; Vos, R.C.H. de; Vervoort, J.J.M. ; Bino, R.J. - \ 2014
Analytical Chemistry 86 (2014)10. - ISSN 0003-2700 - p. 4767 - 4774.
human fecal microbiota - mass-spectrometry - structural elucidation - human plasma - phenolic-compounds - spectral trees - polyphenols - identification - absorption - metabolomics
The colonic breakdown and human biotransformation of small molecules present in food can give rise to a large variety of potentially bioactive metabolites in the human body. However, the absence of reference data for many of these components limits their identification in complex biological samples, such as plasma and urine. We present an in silico workflow for automatic chemical annotation of metabolite profiling data from liquid chromatography coupled with multistage accurate mass spectrometry (LC-MSn), which we used to systematically screen for the presence of tea-derived metabolites in human urine samples after green tea consumption. Reaction rules for intestinal degradation and human biotransformation were systematically applied to chemical structures of 75 green tea components, resulting in a virtual library of 27¿245 potential metabolites. All matching precursor ions in the urine LC–MSn data sets, as well as the corresponding fragment ions, were automatically annotated by in silico generated (sub)structures. The results were evaluated based on 74 previously identified urinary metabolites and lead to the putative identification of 26 additional green tea-derived metabolites. A total of 77% of all annotated metabolites were not present in the Pubchem database, demonstrating the benefit of in silico metabolite prediction for the automatic annotation of yet unknown metabolites in LC–MSn data from nutritional metabolite profiling experiments.
Genetic mapping of semi-polar metabolites in pepper fruits (Capsicum sp.): towards unravelling the molecular regulation of flavonoid quantitative trait loci
Wahyuni, Y. ; Stahl-Hermes, V. ; Ballester, A.R. ; Vos, C.H. de; Voorrips, R.E. ; Maharijaya, A. ; Molthoff, J.W. ; Víquez Zamora, A.M. ; Sudarmonowati, E. ; Arisi, A.C.M. ; Bino, R.J. ; Bovy, A.G. - \ 2014
Molecular Breeding 33 (2014)3. - ISSN 1380-3743 - p. 503 - 518.
l. var. acuminatum - tomato fruit - frankliniella-occidentalis - capsaicinoid content - mass-spectrometry - annuum - expression - metabolomics - glycosides - biodiversity
Untargeted LCMS profiling of semi-polar metabolites followed by metabolite quantitative trait locus (mQTL) analysis was performed in ripe pepper fruits of 113 F2 plants derived from a cross between Capsicum annuum AC1979 (no. 19) and Capsicum chinense No. 4661 Selection (no. 18). The parental accessions were selected based on their variation in fruit morphological characteristics and fruit content of some target phytonutrients. Clear segregation of fruit colour and fruit metabolite profiles was observed in the F2 population. The F2 plants formed three clusters based on their metabolite profiles. Of the total of 542 metabolites, 52 could be annotated, including a range of flavonoids, such as flavone C-glycosides, flavonol O-glycosides and naringenin chalcone, as well as several phenylpropanoids, a capsaicin analogue, fatty acid derivatives and amino acid derivatives. Interval mapping revealed 279 mQTLs in total. Two mQTL hotspots were found on chromosome 9. These two chromosomal regions regulated the relative levels of 35 and 103 metabolites, respectively. Analysis also revealed an mQTL for a capsaicin analogue, located on chromosome 7. Confirmation of flavonoid mQTLs using a set of six flavonoid candidate gene markers and their corresponding expression data (expression QTLs) indicated the Ca-MYB12 transcription factor gene on chromosome 1 and the gene encoding flavone synthase (FS-2) on chromosome 6 as likely causative genes determining the variation in naringenin chalcone and flavone C-glycosides, respectively, in this population. The combination of large-scale metabolite profiling and QTL analysis provided valuable insight into the genomic regions and genes important for the production of (secondary) metabolites in pepper fruit. This will impact breeding strategies aimed at optimising the content of specific metabolites in pepper fruit
Secondary Metabolites of Capsicum Species and Their Importance in the Human Diet
Wahyuni, Y. ; Ballester, A.R. ; Sudarmonowati, E. ; Bino, R.J. ; Bovy, A.G. - \ 2013
Journal of Natural Products 76 (2013)4. - ISSN 0163-3864 - p. 783 - 793.
chlorophyll catabolism pathway - capsaicinoid-like substances - tandem mass-spectrometry - red-pepper paprika - mature fruit color - annuum-l cultivars - cv ch-19 sweet - antioxidant activity - candidate gene - ascorbic-acid
The genus Capsicum (pepper) comprises a large number of wild and cultivated species. The plants are grown all over the world, primarily in tropical and subtropical countries. The fruits are an excellent source of health-related compounds, such as ascorbic acid (vitamin C), carotenoids (provitamin A), to copherols (vitamin E), flavonoids, and capsaicinoids. Pepper fruits have been used for fresh and cooked consumption, as well as for medicinal purposes, such as treatment of asthma, coughs, sore throats, and toothache. Depending on its uses, there are several main characters important for product quality; pungency, bright attractive colors, highly concentrated extracts, and a small number of seeds are the main characters on which quality is based and priced. Herein, a general overview of biochemical composition, medical properties of these compounds, and characteristics of quality attributes of pepper fruits is presented.
|The Large Scale Identification and Quantification of Conjugates of Intact and Gut Microbial Bioconversion Products of Polyphenols.
Hooft, J.J.J. van der; Vos, C.H. de; Bino, R.J. ; Mihaleva, V.V. ; Ridder, L.O. ; Roo, N. de; Jacobs, D.M. ; Duynhoven, J.P.M. van; Vervoort, J. - \ 2013
In: Magnetic Resonance in Food Science / Duynhoven, J., Belton, P.S., Webb, G.A., As, H., CBSG/NMC - ISBN 9781849736343 - p. 177 - 182.
A human diet containing a significant amount of flavonoids, such as present in tea, red wine, apple, and cocoa has been associated with reduced disease risks. After consumption, a part of these flavonoids can be directly absorbed by the small intestine, but the greatest part passages towards the large intestine where microbes break the flavonoids down into phenolic metabolites. After absorption into the blood, both intact and metabolized flavonoids are subsequently methylated, sulphated, and glucuronidated or a combination thereof. The exact chemical structural elucidation and quantification of these conjugates present in the human body are key to identify potential bioactive components. However, this is still a tedious task due to their relative low abundance in a complex background of other high-abundant metabolites and the many possible isomeric forms. Therefore, we aimed to systematically identify these conjugates by using a combination of pre-concentration and separation by solid phase extraction (SPE) followed by LC-FTMSn and 1D 1H NMR. The combination of LC-FTMSn and HPLC-TOF-MS-SPE-NMR resulted in the efficient identification and quantification of low abundant polyphenol metabolites down to micromolar concentrations and thus opens up new perspectives for in depth studying of the bioavailability and the possible mode of action of flavonoids like flavan-3-ols and their gut-microbial break-down products circulating in the human body.
Automatic Chemical Structure Annotation of an LC-MSn Based Metabolic Profile from Green Tea
Ridder, L.O. ; Hooft, J.J.J. van der; Verhoeven, S. ; Vos, C.H. de; Bino, R.J. ; Vervoort, J. - \ 2013
Analytical Chemistry 85 (2013)12. - ISSN 0003-2700 - p. 6033 - 6040.
accurate mass-spectrometry - camelia-sinensis extracts - spectral trees - oolong tea - identification - fragmentation - elucidation - flavan-3-ols - polyphenols - software
Liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSn) can generate comprehensive spectral information of metabolites in crude extracts. To support structural characterization of the many metabolites present in such complex samples, we present a novel method (http://www.emetabolomics.org/magma) to automatically process and annotate the LC–MSn data sets on the basis of candidate molecules from chemical databases, such as PubChem or the Human Metabolite Database. Multistage MSn spectral data is automatically annotated with hierarchical trees of in silico generated substructures of candidate molecules to explain the observed fragment ions and alternative candidates are ranked on the basis of the calculated matching score. We tested this method on an untargeted LC–MSn (n = 3) data set of a green tea extract, generated on an LC-LTQ/Orbitrap hybrid MS system. For the 623 spectral trees obtained in a single LC–MSn run, a total of 116¿240 candidate molecules with monoisotopic masses matching within 5 ppm mass accuracy were retrieved from the PubChem database, ranging from 4 to 1327 candidates per molecular ion. The matching scores were used to rank the candidate molecules for each LC–MSn component. The median and third quartile fractional ranks for 85 previously identified tea compounds were 3.5 and 7.5, respectively. The substructure annotations and rankings provided detailed structural information of the detected components, beyond annotation with elemental formula only. Twenty-four additional components were putatively identified by expert interpretation of the automatically annotated data set, illustrating the potential to support systematic and untargeted metabolite identification.
Structural elucidation of low abundant metabolites in complex sample matrices
Hooft, J.J.J. van der; Vos, R.C.H. de; Ridder, L.O. ; Vervoort, J. ; Bino, R.J. - \ 2013
Metabolomics 9 (2013)5. - ISSN 1573-3882 - p. 1009 - 1018.
ms-spe-nmr - nuclear-magnetic-resonance - solid-phase extraction - liquid-chromatography - spectral trees - circular-dichroism - plant metabolomics - crude extracts - mass-spectra - identification
Identification of metabolites is a major challenge in biological studies and relies in principle on mass spectrometry (MS) and nuclear magnetic resonance (NMR) methods. The increased sensitivity and stability of both NMR and MS systems have made dereplication of complex biological samples feasible. Metabolic databases can be of help in the identification process. Nonetheless, there is still a lack of adequate spectral databases that contain high quality spectra, but new developments in this area will assist in the (semi-)automated identification process in the near future. Here, we discuss new developments for the structural elucidation of low abundant metabolites present in complex sample matrices. We describe how a recently developed combination of high resolution MS multistage fragmentation (MSn) and high resolution one dimensional (1D)-proton (1H)-NMR of liquid chromatography coupled to solid phase extraction (LC–SPE) purified metabolites can circumvent the need for isolating extensive amounts of the compounds of interest to elucidate their structures. The LC–MS–SPE–NMR hardware configuration in conjunction with high quality databases facilitates complete structural elucidation of metabolites even at sub-microgram levels of compound in crude extracts. However, progress is still required to optimally exploit the power of an integrated MS and NMR approach. Especially, there is a need to improve and expand both MSn and NMR spectral databases. Adequate and user-friendly software is required to assist in candidate selection based on the comparison of acquired MS and NMR spectral information with reference data. It is foreseen that these focal points will contribute to a better transfer and exploitation of structural information gained from diverse analytical platforms
Metabolomics and molecular marker analysis to explore pepper (Capsicum sp.) biodiversity
Wahyuni, Y. ; Ballester, A.R. ; Tikunov, Y.M. ; Vos, C.H.R. de; Pelgrom, K.T.B. ; Maharijaya, A. ; Sudarmonowati, E. ; Bino, R.J. ; Bovy, A.G. - \ 2013
Metabolomics 9 (2013)1. - ISSN 1573-3882 - p. 130 - 144.
acyclic diterpene glycosides - annuum l. - capsaicinoid content - mass-spectrometry - volatile fraction - hs-spme - fruit - tomato - chinense - constituents
An overview of the metabolic diversity in ripe fruits of a collection of 32 diverse pepper (Capsicum sp.) accessions was obtained by measuring the composition of both semi-polar and volatile metabolites in fruit pericarp, using untargeted LC–MS and headspace GC–MS platforms, respectively. Accessions represented C. annuum, C. chinense, C. frutescens and C. baccatum species, which were selected based on variation in morphological characters, pungency and geographic origin. Genotypic analysis using AFLP markers confirmed the phylogenetic clustering of accessions according to Capsicum species and separated C. baccatum from the C. annuum–C. chinense–C. frutescens complex. Species-specific clustering was also observed when accessions were grouped based on their semi-polar metabolite profiles. In total 88 semi-polar metabolites could be putatively identified. A large proportion of these metabolites represented conjugates of the main pepper flavonoids (quercetin, apigenin and luteolin) decorated with different sugar groups at different positions along the aglycone. In addition, a large group of acyclic diterpenoid glycosides, called capsianosides, was found to be highly abundant in all C. annuum genotypes. In contrast to the variation in semi-polar metabolites, the variation in volatiles corresponded well to the differences in pungency between the accessions. This was particularly true for branched fatty acid esters present in pungent accessions, which may reflect the activity through the acyl branch of the metabolic pathway leading to capsaicinoids. In addition, large genetic variation was observed for many well-established pepper aroma compounds. These profiling data can be used in breeding programs aimed at improving metabolite-based quality traits such as flavour and health-related metabolites in pepper fruits.
Structural annotation and elucidation of conjugated phenolic compounds in black, green, and white tea extracts
Hooft, J.J.J. van der; Akermi, M. ; Yelda Ünlü, F. ; Mihaleva, V. ; Gomez Roldan, V. ; Bino, R.J. ; Vos, R.C.H. de; Vervoort, J.J.M. - \ 2012
Journal of Agricultural and Food Chemistry 60 (2012)36. - ISSN 0021-8561 - p. 8841 - 8850.
pu-erh tea - mass-spectrometry - oolong tea - hplc-dad - lc-msn - liquid-chromatography - chlorogenic acids - camellia-sinensis - purine alkaloids - ellagic acid
Advanced analytical approaches consisting of both LC-LTQ-Orbitrap Fourier transformed (FT)-MS and LC-time-of-flight-(TOF)-MS coupled to solid-phase extraction (SPE) NMR were used to obtain more insight into the complex phenolic composition of tea. On the basis of the combined structural information from (i) accurate mass fragmentation spectra, derived by using LC-Orbitrap FTMS(n), and (ii) proton NMR spectra, derived after LC-TOFMS triggered SPE trapping of selected compounds, 177 phenolic compounds were annotated. Most of these phenolics were glycosylated and acetylated derivatives of flavan-3-ols and flavonols. Principal component analysis based on the relative abundance of the annotated phenolic compounds in 17 commercially available black, green, and white tea products separated the black teas from the green and white teas, with epicatechin-3,5-di-O-gallate and prodelphinidin-O-gallate being among the main discriminators. The results indicate that the combined use of LC-LTQ-Orbitrap FTMS and LC-TOFMS-SPE-NMR leads to a more comprehensive metabolite description and comparison of tea and other plant samples
Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga
Lamers, P.P. ; Janssen, M. ; Vos, R.C.H. de; Bino, R.J. ; Wijffels, R.H. - \ 2012
Journal of Biotechnology 162 (2012)1. - ISSN 0168-1656 - p. 21 - 27.
beta-carotene - biochemical-composition - nutrient limitation - low-temperature - high light - accumulation - bardawil - alga - induction - stress
Nitrogen availability and light intensity affect ß-carotene overproduction in the green alga Dunaliella salina. Following a previous study on high-light stress, we here report on the effect of nitrogen depletion on the growth characteristics and ß-carotene as well as fatty acid metabolism of D. salina under a constant light regime in a turbidostat. Upon nitrogen depletion, the biomass yield on absorbed light approximately doubled, due to a transient increase in cell division rate, swelling of the cells and a linear increase of the density of the cells. Simultaneously, ß-carotene started to accumulate up to a final intracellular concentration of 14 mg LCV-1 (i.e. 2.7% of AFDW). This ß-carotene production accounted for 6% of the increased density of the cells, indicating that other biochemical constituents accumulated as well. Since D. salina accumulates ß-carotene in lipid globules, we also determined the fatty acid content and composition of D. salina. The intracellular concentration of the total fatty acid pool did not change significantly during nitrogen starvation, indicating that ß-carotene and total fatty acid accumulation were unrelated, similar to what was found previously for high-light treated cells. However, for both high-light and nitrogen stress, ß-carotene accumulation negatively correlated with the degree of unsaturation of the total fatty acid pool and, within the individual fatty acids, correlated positively with oleic acid biosynthesis, suggesting that oleic acid may be a key component of the lipid-globule-localized triacylglycerols and thereby in ß-carotene accumulation.
Structural elucidation and quantification of phenolic conjugates present in human urine after tea intake
Hooft, J.J.J. van der; Vos, R.C.H. de; Mihaleva, V. ; Bino, R.J. ; Ridder, L.O. ; Roo, N. de; Jacobs, D.M. ; Duynhoven, J.P.M. van; Vervoort, J.J.M. - \ 2012
Analytical Chemistry 84 (2012)16. - ISSN 0003-2700 - p. 7263 - 7271.
tandem mass-spectrometry - green tea - black tea - metabolite identification - ellagic acid - metabolomics - polyphenols - nmr - ingestion - phytochemicals
In dietary polyphenol exposure studies, annotation and identification of urinary metabolites present at low (micromolar) concentrations are major obstacles. In order to determine the biological activity of specific components, it is necessary to have the correct structures and the quantification of the polyphenol-derived conjugates present in the human body. We present a procedure for identification and quantification of metabolites and conjugates excreted in human urine after single bolus intake of black or green tea. A combination of a solid phase extraction (SPE) preparation step and two high pressure liquid chromatography (HPLC)-based analytical platforms was used; namely, accurate mass fragmentation (HPLC-FTMSn) and mass-guided SPE-trapping of selected compounds for nuclear magnetic resonance spectroscopy (NMR) measurements (HPLC-TOFMS-SPE-NMR). HPLC-FTMSn analysis led to the annotation of 138 urinary metabolites, including 48 valerolactone and valeric acid conjugates. By combining the results from MSn fragmentation with the one dimensional (1D)-1H-NMR spectra of HPLC-TOFMS-SPE trapped compounds, we elucidated the structures of 36 phenolic conjugates, including the glucuronides of 3’,4’-di, and 3’,4’,5’-trihydroxyphenyl-¿-valerolactone, three urolithin glucuronides, and indole-3-acetic acid glucuronide. We also obtained 26 hours of quantitative excretion profiles for specific valerolactone conjugates. The combination of the HPLC-FTMSn and HPLC-TOFMS-SPE-NMR platforms results in the efficient identification and quantification of low abundant phenolic conjugates down to nanomoles of trapped amounts of metabolite corresponding to micromolar metabolite concentrations in urine
Characterization of the natural variation in Arabidopsis thaliana metabolome by the analysis of metabolic distance
Houshyani Hassanzadeh, B. ; Kabouw, P. ; Muth, D. ; Vos, R.C.H. de; Bino, R.J. ; Bouwmeester, H.J. - \ 2012
Metabolomics 8 (2012)suppl. 1. - ISSN 1573-3882 - p. 131 - 145.
genomic diversity - mass-spectrometry - functional genomics - mildew resistance - plants - tomato - biosynthesis - pathogens - volatiles - defense
Metabolite fingerprinting is widely used to unravel the chemical characteristics of biological samples. Multivariate data analysis and other statistical tools are subsequently used to analyze and visualize the plasticity of the metabolome and/or the relationship between those samples. However, there are limitations to these approaches for example because of the multi-dimensionality of the data that makes interpretation of the data obtained from untargeted analysis almost impossible for an average human being. These limitations make the biological information that is of prime importance in untargeted studies be partially exploited. Even in the case of full exploitation, current methods for relationship elucidation focus mainly on between groups variation and differences. Therefore, a measure that is capable of exploiting both between- and within-group biological variation would be of great value. Here, we examined the natural variation in the metabolome of nine Arabidopsis thaliana accessions grown under various environmental conditions and established a measure for the metabolic distance between accessions and across environments. This data analysis approach shows that there is just a minor correlation between genetic and metabolic diversity of the nine accessions. On the other hand, it delivers so far in Arabidopsis unexplored chemical information and is shown to be biologically relevant for resistance studies.
Spectral trees as a robust annotation tool in LC–MS based metabolomics
Hooft, J.J.J. van der; Vervoort, J.J.M. ; Bino, R.J. ; Vos, C.H. de - \ 2012
Metabolomics 8 (2012)4. - ISSN 1573-3882 - p. 691 - 703.
orbitrap mass-spectrometry - liquid-chromatography - tomato fruit - secondary metabolites - phenolic-compounds - brassica-rapa - hplc-dad - identification - flavonoids - arabidopsis
The identification of large series of metabolites detectable by mass spectrometry (MS) in crude extracts is a challenging task. In order to test and apply the so-called multistage mass spectrometry (MS n ) spectral tree approach as tool in metabolite identification in complex sample extracts, we firstly performed liquid chromatography (LC) with online electrospray ionization (ESI)–MS n , using crude extracts from both tomato fruit and Arabidopsis leaf. Secondly, the extracts were automatically fractionated by a NanoMate LC-fraction collector/injection robot (Advion) and selected LC-fractions were subsequently analyzed using nanospray-direct infusion to generate offline in-depth MS n spectral trees at high mass resolution. Characterization and subsequent annotation of metabolites was achieved by detailed analysis of the MS n spectral trees, thereby focusing on two major plant secondary metabolite classes: phenolics and glucosinolates. Following this approach, we were able to discriminate all selected flavonoid glycosides, based on their unique MS n fragmentation patterns in either negative or positive ionization mode. As a proof of principle, we report here 127 annotated metabolites in the tomato and Arabidopsis extracts, including 21 novel metabolites. Our results indicate that online LC–MS n fragmentation in combination with databases of in-depth spectral trees generated offline can provide a fast and reliable characterization and annotation of metabolites present in complex crude extracts such as those from plants.
A strategy for fast structural elucidation of metabolites in small volume plant extracts using automated MS-guided LC-MS-SPE-NMR
Hooft, J.J.J. van der; Mihaleva, V.V. ; Vos, R.C.H. de; Bino, R.J. ; Vervoort, J.J.M. - \ 2011
Magnetic Resonance in Chemistry 49 (2011)Suppl. S1. - ISSN 0749-1581 - p. S55 - S60.
nuclear-magnetic-resonance - solid-phase extraction - natural-products research - liquid-chromatography - mass-spectrometry - flavonoids - tomato - identification - metabolomics - hyphenation
Fast and reliable metabolite identification based on automated MS-guided HPLC-MS-SPE-NMR metabolite extraction combined with an automated 1H NMR spectrum fitting was developed. Positional isomers as structure 1 and 2 were easily distinguished. In many metabolomics studies, metabolite identification by mass spectrometry (MS) often is hampered by the lack of good reference compounds, and hence, NMR information is essential for structural elucidation, especially for the very large group of secondary metabolites. The classical approach for compound identification is to perform time-consuming and laborious HPLC fractionations and purifications, before (re)dissolving the molecules in deuterated solvents for NMR measurements. Hence, a more direct and easy purification protocol would save time and efforts. Here, we propose an automated MS-guided HPLC-MS-solid phase extraction-NMR approach, which was used to fully characterize flavonoid structures present in crude tomato plant extracts. NMR spectra of plant metabolites, automatically trapped and purified from LC-MS traces, were successfully obtained, leading to the structural elucidation of the metabolites. The MS-based trapping enabled a direct link between the mass signals and NMR peaks derived from the selected LC-MS peaks, thereby decreasing the time needed for elucidation of the metabolite structures. In addition, automated 1H NMR spectrum fitting further speeded up the candidate rejection process. Our approach facilitates the more rapid unraveling of yet unknown metabolite structures and can therefore make untargeted metabolomics approaches more powerful
Metabolite biodiversity in pepper (Capsicum) fruits of thirty-two diverse accessions : variation in health-related compounds and impliciations for breeding
Wahyuni, Y. ; Ballester, A.R. ; Sudarmonowati, E. ; Bino, R.J. ; Bovy, A.G. - \ 2011
Phytochemistry 72 (2011)11-12. - ISSN 0031-9422 - p. 1358 - 1370.
capsanthin-capsorubin synthase - annuum-l - ascorbic-acid - liquid-chromatography - capsaicinoid analogs - antioxidant activity - carotenoid-pigments - mass-spectrometry - candidate gene - ch-19 sweet
A comprehensive study on morphology and biochemical compounds of 32 Capsicum spp. accessions has been performed. Accessions represented four pepper species, Capsicum annuum, Capsicum frutescens, Capsicum chinense and Capsicum baccatum which were selected by their variation in morphological characters such as fruit color, pungency and origin. Major metabolites in fruits of pepper, carotenoids, capsaicinoids (pungency), flavonoid glycosides, and vitamins C and E were analyzed and quantified by high performance liquid chromatography. The results showed that composition and level of metabolites in fruits varied greatly between accessions and was independent of species and geographical location. Fruit color was determined by the accumulation of specific carotenoids leading to salmon, yellow, orange, red and brown colored fruits. Levels of both O- and C-glycosides of quercetin, luteolin and apigenin varied strongly between accessions. All non-pungent accessions were devoid of capsaicins, whereas capsaicinoid levels ranged from 0.07 up to 80 mg/100 g fr. wt. in fruit pericarp. In general, pungent accessions accumulated the highest capsaicinoid levels in placenta plus seed tissue compared to pericarp. The non-pungent capsaicinoid analogs, capsiates, could be detected at low levels in some pungent accessions. All accessions accumulated high levels of vitamin C, up to 200 mg/100 g fr. wt. The highest vitamin E concentration found was 16 mg/100 g fr. wt. Based on these metabolic data, five accessions were selected for further metabolic and molecular analysis, in order to isolate key genes involved in the production of these compounds and to assist future breeding programs aimed at optimizing the levels of health-related compounds in pepper fruit.
Bino, Raoul - \ 2011
Polyphenol Identification Based on Systematic and Robust High-Resolution Accurate Mass Spectroscopy Fragmentation
Hooft, J.J.J. van der; Vervoort, J.J.M. ; Bino, R.J. ; Beekwilder, M.J. ; Vos, R.C.H. de - \ 2011
Analytical Chemistry 83 (2011)1. - ISSN 0003-2700 - p. 409 - 416.
performance liquid-chromatography - negative electrospray-ionization - collision-induced dissociation - nuclear-magnetic-resonance - flavonoid glycosides - phenolic-compounds - structural-characterization - plant metabolomics - o-glycosides - msn
High-mass resolution multi-stage mass spectrometry (MSn) fragmentation was tested for differentiation and identification of metabolites, using a series of 121 polyphenolic molecules. The MSn fragmentation approach is based on the systematic breakdown of compounds, forming a so-called spectral tree. A chip-based nanoelectrospray ionization source was used combined with an ion-trap, providing reproducible fragmentation, and accurate mass read-out in an Orbitrap Fourier transform (FT) MS enabling rapid assignment of elemental formulas to the molecular ions and all fragment ions derived thereof. The used protocol resulted in reproducible MSn fragmentation trees up to MS5. Obtained results were stable over a 5 month time period, a concentration change of 100-fold, and small changes in normalized collision energy, which is key to metabolite annotation and helpful in structure and substructure elucidation. Differences in the hydroxylation and methoxylation patterns of polyphenolic core structures were found to be reflected by the differential fragmentation of the entire molecule, while variation in a glycosylation site displayed reproducible differences in the relative intensities of fragments originating from the same aglycone fragment ion. Accurate MSn-based spectral tree data are therefore a powerful tool to distinguish metabolites with similar elemental formula, thereby assisting compound identification in complex biological samples such as crude plant extracts.