Estratègies cromatogràfiques i quimiomètriques per a estudis de metabolòmica no dirigida en arròs (Oryza sativa L.)

[cat] Les mostres analitzades en els estudis de metabolòmica ambiental presenten una elevada complexitat per la gran quantitat d’informació bioquímica que contenen, especialment, en el cas d’estudis no dirigits. En aquests estudis és necessari l’ús de tècniques analítiques d’alt rendiment com, per exemple, la cromatografia de líquids acoblada a espectrometria de masses (LC-MS), les quals permeten analitzar els canvis en la concentració dels metabòlits en diferents tipus de mostres. D’aquesta forma es generen grans conjunts de dades multivariants (amb desenes de mostres i milers de variables), els quals requereixen l’aplicació d’eines quimiomètriques d’anàlisi de dades per tal d’extreure la informació d’interès. En aquesta Tesi s’han desenvolupat i aplicat nous mètodes d’anàlisi basats en tècniques de cromatografia de líquids uni- i bidimensional acoblada a espectrometria de masses, conjuntament amb noves estratègies d’anàlisi multivariant de dades que permeten extreure la màxima informació bioquímica en estudis de metabolòmica no dirigida d’organismes vegetals. En un estudi metabolòmic no dirigit, els reptes analítics i quimiomètrics més importants són: l’anàlisi instrumental exhaustiva del màxim nombre de metabòlits presents a les mostres, l’anàlisi quimiomètrica de les dades experimentals generades i la identificació i quantificació dels metabòlits resolts, especialment d’aquells que mostren canvis importants en la seva concentració degut a l’estímul o estrès aplicat sobre les mostres analitzades. En aquesta Tesi s’han avaluat i proposat diverses estratègies i eines que poden resultar útils per solucionar els reptes existents en aquestes etapes. S’ha estudiat per exemple la influència que poden tenir diferents factors experimentals en la separació dels metabòlits mitjançant cromatografia d’interacció hidrofílica (HILIC) com ara el tipus de fase estacionària, el pH i la força iònica de la fase mòbil. Els resultats obtinguts han demostrat que els factors més importants són el tipus de fase estacionària HILIC i el pH de la fase mòbil. Concretament, en l’estudi dels diferents tipus de fase estacionària s’ha determinat que les fases amida i zwitteriònica són les que proporcionen millors resultats per a l’anàlisi de metabòlits polars. D’altra banda, s’han comparat dues estratègies de tractament de dades de metabolòmica no dirigida: l’estratègia de referència en el camp basada en la utilització del programa XCMS, i la que s’ha proposat en aquesta Tesi, que es basa en el mètode quimiomètric de resolució multivariant de corbes per mínims quadrats alternats (MCR-ALS). Els dos procediments han proporcionat resultats similars i, en conseqüència, tots dos resulten apropiats pel tractament de dades de metabolòmica no dirigida. Però cal destacar que el procediment basat en MCR-ALS és més robust ja que s’ha pogut aplicar a una varietat major de dades experimentals. S’ha proposat també l’ús de models de relació quantitativa estructura-retenció (QSRR) que han permès relacionar l’estructura dels metabòlits amb el seu temps de retenció observat experimentalment. Els resultats obtinguts demostren que l’aplicació de models QSRR pot ser una eina a considerar per a la identificació dels metabòlits, ja que redueix el nombre de possibles candidats i en facilita la seva confirmació. La complexitat de les mostres analitzades en els estudis metabolòmics sovint excedeix els límits de capacitat de pic aconseguits per la cromatografia de líquids unidimensional. Per aquest motiu, la cromatografia de líquids bidimensional exhaustiva (LC×LC) es presenta com una bona tècnica alternativa que permet ampliar la capacitat de separació de mescles molt complexes, com ara les d’extractes de metabòlits de mostres biològiques. S’ha avaluat l’estructura de les dades obtingudes a partir de l’aplicació de LC×LC-MS, i la seva possible modelització. Els resultats obtinguts han mostrat que, en general, el model de MCR-ALS bilineal és el més indicat per a l’anàlisi d’aquest tipus de dades. S’ha estudiat també el possible ús del procediment ROIMCR (compressió de les dades per cerca de regions d’interès [ROIs] seguida d’una resolució mitjançant MCR-ALS) per a estudis de metabolòmica no dirigida mitjançant de LC×LC-MS. Els resultats han mostrat que la combinació de les tècniques de separació multidimensional i dels mètodes quimiomètrics de resolució és adequada en estudis metabolòmics, ja que permet resoldre i identificar un nombre molt elevat de metabòlits i lípids en una sola anàlisi. Finalment, s’ha investigat la influència de diferents factors estressants ambientals sobre el metaboloma d’un organisme vegetal model com l’arròs (Oryza sativa L.). Els factors estressants ambientals estudiats han estat abiòtics, com ara, l’estrès hídric, l’augment de la temperatura i la presència de metalls pesants (Cd, Cu i As) contaminants al sòl. Els resultats obtinguts han demostrat que els tres factors ambientals avaluats afecten de forma significativa el metaboloma de l’arròs durant el seu creixement. Els espectres de masses resolts s’han utilitzat per a la identificació dels metabòlits i lípids de les mostres biològiques analitzades. La interpretació biològica dels canvis de concentració dels metabòlits i lípids identificats han demostrat que la contaminació per metalls pesants ocasiona una disminució del creixement i de l’activitat fotosintètica de la planta, i que s’indueix un mecanisme de desintoxicació que permet disminuir el dany cel·lular. S’ha demostrat també que els increments de temperatura i d’estrès hídric provoquen un reajustament important del grau d’insaturació dels lípids de les cèl·lules de l’arròs, el qual permet millorar de forma considerable la fluïdesa de les membranes cel·lulars.[eng] In environmental metabolomic studies, the analysed samples are highly complex due to the great amount of biochemical information contained therein, especially in the case of non-targeted studies. In these studies, it is necessary to use high-throughput analytical methodologies, such as liquid chromatography coupled with mass spectrometry (LC-MS), which allow analysing the concentration changes of metabolites among different types of samples. Multivariate datasets (with dozens of samples and thousands of variables) are generated in order to retrieve the information of interest. These huge datasets required the application of chemometric tools during the data treatment. In this Thesis, new analytical methods based on mono- and two-dimensional liquid chromatography coupled with mass spectrometry, combined with new multivariate data analysis strategies, have been developed and applied to obtain the major biochemical information in non-targeted metabolomic studies of vegetal organisms. In a non-targeted metabolomic study, the most important analytical and chemometric challenges are: instrumental analysis of metabolites, chemometric analysis of the experimental generated data and identification of metabolites, especially of those that show significant changes in their concentration due to the stress applied to the analysed samples. In this Thesis, different tools and strategies that can help to overcome these challenges have been proposed and assessed. For instance, the influence of different experimental factors in the separation of metabolites by hydrophilic chromatographic interaction (HILIC), such as the type of stationary phase, the pH and the ionic strength of the mobile phase, has been studied. The obtained results have shown that the most important factors are the type of stationary phase and the pH of the mobile phase. Specifically, in the comparison of different stationary phases, the amide and zwitterionic phases have been determined to provide better results for the analysis of polar metabolites. On the other hand, two data treatment strategies for non-targeted metabolomics data have been compared: the approach based on the XCMS software (which is the standard strategy in the metabolomics field) and the approach based on the multivariate curve resolution by alternating least squares (MCR-ALS) method (which has been proposed in the presented Thesis). Both procedures have provided similar results, and consequently, both are appropriate for the treatment of non-targeted metabolomic data. The MCR-ALS based procedure is more robust since it has been applied to a greater variety of experimental data. Moreover, the use of quantitative structure-retention relationship (QSRR) models, which have been able to relate the structure of the metabolites with their experimentally observed retention time, has also been proposed. The obtained results showed that the applications of QSRR models can be a useful tool for the identification of metabolites since it reduces the number of possible candidates and facilitates their confirmation. The complexity of the samples analysed in metabolomics studies often exceed the limits of peak capacity achievable by one-dimensional liquid chromatography. For this reason, comprehensive two- dimensional liquid chromatography (LC×LC) appears as a good alternative that increases the separation capacities of highly complex samples, such as extracts of metabolites from biological samples. The structure of the data obtained from LC×LC-MS analysis and its possible modelling have been evaluated. The obtained results have shown that generally, the MCR-ALS bilinear model is the most suitable for the analysis of this type of data. The use of ROIMCR data treatment approach (data compression by the search of regions of interest [ROI] and data resolution using MCR-ALS) in non-targeted metabolomics studies using LC×LC-MS has been studied. Results have shown that the combination of multidimensional separation techniques and chemometric methods is properly adequate in non-targeted metabolomic studies, since it allows the resolution and identification of a great number of metabolites in a single analysis. Finally, the effect of different environmental stressors on the metabolome of a vegetal model organism such as rice (Oryza sativa L.) has been investigated. The studied environmental stressors have been abiotic, such as hydric stress, temperature alterations and heavy metal (Cd, Cu and As) pollution. The obtained results have demonstrated that the three evaluated environmental factors significantly alter the rice metabolome. The resolved mass spectra have been used to identify the lipids and metabolites contained in the analysed rice samples. The biological interpretation of concentration changes of the identified lipids and metabolites have shown on the one hand that heavy metal pollution causes a decrease in growth and photosynthetic activity of the plant, and also it induces a detoxification mechanism that reduces cell damage. On the other hand, it has also been demonstrated that increases in temperature and hydric stress causes an important readjustment of the unsaturation degree of lipids in rice cells, which improve the fluidity of cell membranes

Characterization of fruit products by capillary zone electrophoresis and liquid chromatography using the compositional profiles of polyphenols. Application to authentication of natural extracts

Capillary zone electrophoresis (CZE) and high performance liquid chromatography (HPLC) were applied to the authentication of fruit products based on the compositional profiles of polyphenols. Various sample treatments were used to maximize the overall recovery of polyphenols or specific fractions, such as phenolic acids or anthocyanins. The resulting CZE and HPLC data were treated with Principal Component Analysis (PCA) showing that samples were mainly clustered according to the fruit of origin, with cranberry- and grape-based products clearly separated in groups. A possible adulterated cranberry extract was analyzed more deeply by high resolution mass spectrometry (HRMS) in order to identify the presence of A-type proanthocyanidins, which are characteristic and more abundant in cranberry-based products. In accordance with PCA interpretation, HRMS results indicated that the suspicious sample was not a cranberry-based product, allowing us to validate and demonstrate the suitability of both CZE- and HPLC-proposed methods for the characterization of fruit-based products

Determination of polyphenolic profiles by liquid chromatography-electrospray-tandem mass spectrometry for the authentication of fruit extracts

Liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) was applied to the analysis and authentication of fruit-based products and fruit-based pharmaceutical preparations. A Kinetex C18 reversed-phase column under gradient elution with 0.1 % formic acid aqueous solution and methanol mobile phases was used for the simultaneous determination of 26 polyphenols, allowing an acceptable separation in less than 22 min. Instrumental quality parameters such as limits of detection (LOD, values between 12-14 µg/L for 19 of the 26 analyzed polyphenols), linearity (r2 > 0.991), run-to-run and day-to-day precisions (RSD values lower than 9.9 and 13.5 %, respectively), and accuracy (relative errors lower than 8 %) were established. A simple extraction method, consisting of a sample sonication with acetone:water:hydrochloric acid (70:29.9:0.1 v/v/v) and centrifugation, was proposed. Two calibration procedures, external calibration using standards prepared in water and standard addition, were evaluated for polyphenol quantification in several grape and cranberry fruits and processed fruit products. For a 95 % confidence level, no statistical differences were observed between the two calibration methods (p values between 0.06 and 0.95), denoting that external calibration was suitable enough for the quantitative analysis of polyphenols in fruit-based products. The proposed LC-ESI-MS/MS method was then applied to the analysis of polyphenols in 23 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic concentration data was then analyzed by principal component analysis (PCA) to extract information of the most significant profile data contributing to authentication of natural extracts according to their fruit of origin

El cultivo del caqui

En este libro se escribe del caqui en toda su amplitud, los mejores investigadores en las diferentes especialidades nos explican los resultados de la investigación y experiencias en fertirrigación, pies y variedades, sanidad vegetal, poda y postcosecha; acompañado y documentado de una amplia bibliografía de los investigadores de los principales países productores del mundo: China, Japón e Israel, e Italia, más que por su producción, por su continua y avanzada investigación. Este libro, fruto del trabajo de las diferentes líneas de investigación del producto, coordinada por el magnífico equipo de investigadores del IVIA, pone a disposición de la producción, postcosecha y comercialización todos los avances conocidos a nivel nacional e internacional

Modelling of Hydrophilic Interaction Liquid Chromatography Stationary Phases Using Chemometric Approaches

Metabolomics is a powerful and widely used approach that aims to screen endogenous small molecules (metabolites) of different families present in biological samples. The large variety of compounds to be determined and their wide diversity of physical and chemical properties have promoted the development of different types of hydrophilic interaction liquid chromatography (HILIC) stationary phases. However, the selection of the most suitable HILIC stationary phase is not straightforward. In this work, four different HILIC stationary phases have been compared to evaluate their potential application for the analysis of a complex mixture of metabolites, a situation similar to that found in non-targeted metabolomics studies. The obtained chromatographic data were analyzed by different chemometric methods to explore the behavior of the considered stationary phases. ANOVA-simultaneous component analysis (ASCA), principal component analysis (PCA) and partial least squares regression (PLS) were used to explore the experimental factors affecting the stationary phase performance, the main similarities and differences among chromatographic conditions used (stationary phase and pH) and the molecular descriptors most useful to understand the behavior of each stationary phase.The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement Number 320737.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

An untargeted lipidomic strategy combining comprehensive two-dimensional liquid chromatography and chemometric analysis

Untargeted lipidomic samples are extremely complex and often exceed the limits of peak capacity achievable by one-dimensional liquid chromatography (LC). Comprehensive two-dimensional liquid chromatography (LC × LC) appears as a promising alternative to overcome this drawback. Unfortunately, this approach generates highly complex datasets which untargeted analysis is challenging. In this work, a global methodological strategy combining LC × LC–MS/MS with chemometric data analysis is proposed for untargeted lipidomic studies. The feasibility of the proposed methodology is demonstrated by its application to assess the effects of arsenic exposure on the lipidome of growing rice samples. A two-dimensional chromatographic setup coupling reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) modes together with a triple quadrupole mass detector (TQD) is proposed to analyze lipid extracts from rice samples at different experimental conditions. Chemometric tools were used for data compression, spectral and elution profiles resolution, feature detection and statistical analysis of the multidimensional LC × LC–MS/MS data. The obtained results revealed that the proposed methodology was useful to gather relevant information from untargeted lipidomic studies and detect potential biomarkers. © 2018 Elsevier B.V.The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement Number 320737Peer reviewe

Chemometric evaluation of hydrophilic interaction liquid chromatography stationary phases: Resolving complex mixtures of metabolites

Different hydrophilic interaction liquid chromatography (HILIC) stationary phases have been evaluated using different chemometric methods with the aim of their application in metabolomics studies. Experimental factors, such as the type of HILIC stationary phase (i.e. amide, amine, zwitterionic and diol) and the mobile phase conditions (organic co-solvent, pH and ionic strength) were assessed using a full factorial experimental design. A test sample mixture of metabolites with diverse physicochemical properties (amino acids, nucleotides, nucleosides, and sugars among others) was analyzed by liquid chromatography with a diode array detector (LC-DAD) using five different HILIC columns. Application of multivariate curve resolution alternating least squares (MCR-ALS) method, allowed the full chromatographic peak resolution of all mixture constituents. This approach was particularly helpful in the case of methanol samples where the quality of the chromatographic separation (resolution) was lower in consequence of the co-solvent perturbation on the water layer formation at the surface of the stationary phase. Then, Berridge chromatographic response function (CRF), based on peak resolution, retention times and number of peaks, was used for the investigation of the best HILIC column configuration for future metabolomics studies. The best chromatographic configuration resulted in being the amide and zwitterionic HILIC stationary phases in combination with acetonitrile as organic co-solvent of the mobile phase. © 2017 The Royal Society of Chemistry.The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement No. 320737. Research funding from MINECO Spain Grant No. CTQ2015-66254-C2-1-P is also acknowledged. The authors are grateful to Ms. Marta Fontal from IDAEA-CSIC (Barcelona, Spain) for technical assistance in the use of the LC-DAD system.Peer reviewe

Untargeted lipidomic evaluation of hydric and heat stresses on rice growth

Environmental stresses are the major factors that limit the geographical distribution of plants. As a consequence, plants have developed different strategies to adapt to these environmental changes among which can be outlined the maintenance of membranes' integrity and fluidity. Lipids are key molecules for this environmental adaptation and a comprehensive understand of the molecular mechanisms underlying is still required. Here, lipidome changes in Japanese rice (Oryza sativa var. Japonica) upon heat and hydric stresses are assessed using an untargeted approach based on liquid chromatography coupled with mass spectrometry (LC-MS). The obtained data were analyzed using different multivariate data analysis tools. A total number of 298 lipids responded to these abiotic stresses, and 128 of them were tentatively identified. Diacylglycerols (DG), triacylglycerols (TG), phosphatidylcholines (PC) and phosphatidylethanolamines (PE) were the most altered lipid families heat and hydric stress. Interpretation of the obtained results showed relevant changes related to the unsaturation degree in the identified lipids. In the case of heat stress, a decrease in the unsaturation degree of lipids can be linked to an increase in the cell membranes' rigidity. In contrast, the hydric stress produced an increase in the lipids unsaturation degree causing an increase in the cell membranes' fluidity, in an attempt to adapt to these non-optimal conditions. © 2018The research leading to these results has received funding from the European Research Council under the European Union Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. 320737 . Also, the authors acknowledge support from both the Ministry of Economy, Industry and Competitiveness (Grant CTQ2017-82598-P ) and the Catalan Agency for Management of University and Research Grants (AGAUR) ( 2017SGR753 ). The authors would like to thank CRAG for kindly supplying Japanese rice seeds.Peer reviewe

Evaluation of changes induced in rice metabolome by Cd and Cu exposure using LC-MS with XCMS and MCR-ALS data analysis strategies

The comprehensive analysis of untargeted metabolomics data acquired using LC-MS is still a major challenge. Different data analysis tools have been developed in recent years such as XCMS (various forms (X) of chromatography mass spectrometry) and multivariate curve resolution alternating least squares (MCR-ALS)-based strategies. In this work, metabolites extracted from rice tissues cultivated in an environmental test chamber were subjected to untargeted full-scan LC-MS analysis, and the obtained data sets were analyzed using XCMS and MCR-ALS. These approaches were compared in the investigation of the effects of copper and cadmium exposure on rice tissue (roots and aerial parts) samples. Both methods give, as a result of their application, the whole set of resolved elution and spectra profiles of the extracted metabolites in control and metal-treated samples, as well as the values of their corresponding chromatographic peak areas. The effects caused by the two considered metals on rice samples were assessed by further chemometric analysis and statistical evaluation of these peak area values. Results showed that there was a statistically significant interaction between the considered factors (type of metal of treatment and tissue). Also, the discrimination of the samples according to both factors was possible. A tentative identification of the most discriminant metabolites (biomarkers) was assessed. It is finally concluded that both XCMS- and MCR-ALS-based strategies provided similar results in all the considered cases despite the completely different approaches used by these two methods in the chromatographic peak resolution and detection strategies. Finally, advantages and disadvantages of using these two methods are discussed. [Figure not available: see fulltext.]Fil: Navarro Reig, Meritxell. Consejo Superior de Investigaciones Científicas; EspañaFil: Jaumot, Joaquim. Consejo Superior de Investigaciones Científicas; EspañaFil: Garcia Reiriz, Alejandro Gabriel. Consejo Superior de Investigaciones Científicas; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Tauler, Romà. Consejo Superior de Investigaciones Científicas; Españ

Metabolomic analysis of the effects of cadmium and copper treatment in: Oryza sativa L. using untargeted liquid chromatography coupled to high resolution mass spectrometry and all-ion fragmentation

While the knowledge of plant metabolomes has increased in the last few years, their response to the presence of toxicants is still poorly understood. Here, we analyse the metabolomic changes in Japanese rice (Oryza sativa var. Japonica) upon exposure to heavy metals (Cd(ii) and Cu(ii)) in concentrations from 10 to 1000 μM. After harvesting, rice metabolites were extracted from aerial parts of the plants and analysed by HPLC (HILIC TSK gel amide-80 column) coupled to a mass spectrometer quadrupole-Orbitrap (Q-Exactive). Full scan and all ion fragmentation (AIF) mass spectrometry modes were used during the analysis. The proposed untargeted metabolomics data analysis strategy is based on the application of the multivariate curve resolution alternating least squares (MCR-ALS) method for feature detection, allowing the simultaneous resolution of pure chromatographic profiles and mass spectra of all metabolites present in the analysed rice extracts. All-ion fragmentation data were used to confirm the identification of MCR-ALS resolved metabolites. A total of 112 metabolites were detected, and 97 of them were subsequently identified and confirmed. Pathway analysis of the observed metabolic changes suggested an underlying similarity of the responses of the plant to Cd(ii) and Cu(ii), although the former treatment appeared to be the more severe of the two. In both cases, secondary metabolism and amino acid-, purine-, carbon- and glycerolipid-metabolism pathways were affected, in a pattern consistent with reduction in plant growth and/or photosynthetic capacity and with induction of defence mechanisms to reduce cell damage. © The Royal Society of Chemistry 2017.The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement no. 320737. The authors would like to thank Center for Research in Agricultural Genomics (CRAG) for kindly supplying Japanese rice seeds.Peer reviewe