UPLC-MS-based metabolite analysis in tomato

Department of Plant Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 860:129-44. DOI: 10.1007/978-1-61779-594-7_9
Source: PubMed


Recent advances in the performance of hyphenated technologies based on ultrapressure chromatography and high-sensitivity mass spectrometry have set the stage for a myriad of metabolomics studies in plants and other organisms. In this chapter, we describe the use of a UPLC (Ultraperformance Liquid Chromatography)-qTOF (quadrupole time-of-flight) system for profiling semipolar metabolites in the model fruit plant tomato. An optimized extraction method, instrument parameters and data treatment procedures are provided. The value of UPLC instruments, which use small particle size chromatographic columns, in terms of resolution, separation, and short injection times are presented. When coupled to a TOF mass spectrometer with high resolution and mass accuracy, good dynamic range, and a fast spectral acquisition capacity, this system is most suitable for the extensive profiling of hundreds of plant metabolites.

  • Source
    • "Future efforts aimed at identifying preserved metabolic processes across fruits from different species would have to include careful experimental design with possibly aligned developmental and ripening states to further test the conclusions from our study. Recent technological advances mean that in excess of 1,000 metabolites can now be readily detected in biological samples (Giavalisco et al., 2011; Rogachev and Aharoni, 2012). Therefore, such an undertaking could additionally be anticipated to cover a larger portion of plant metabolism, including both central and specialized metabolisms (Higashi and Saito, 2013 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Computational analyses of molecular phenotypes traditionally aim at identifying biochemical components that exhibit differential expression under various scenarios (e.g., environmental and internal perturbations) in a single species. High-throughput metabolomics technologies allow quantification of (relative) metabolite levels across developmental stages in different tissues, organs, and species. Novel methods for analyzing the resulting multiple data tables could reveal preserved dynamics of metabolic processes across species. The problem we address in this study is twofold: (i) we derive a single data table, referred to as a compromise, which captures information common to the investigated set of multiple tables containing data on different fruit development and ripening stages in three climacteric (i.e., peach and two tomato cultivars, Ailsa Craig and M82) and two non-climacteric (i.e., strawberry, pepper) fruits; in addition, we demonstrate the power of the method to discern similarities and differences between multiple tables by analyzing publically available metabolomics data from three tomato ripening mutants together with two tomato cultivars, and (ii) identify the conserved dynamics of metabolic processes, reflected in the data profiles of the corresponding metabolites which contribute most to the determined compromise. Our analysis is based on an extension to principle component analysis, called STATIS, in combination with pathway over-enrichment analysis. Based on publically available metabolic profiles for the investigated species, we demonstrate that STATIS can be used to identify the metabolic processes whose behavior is similarly affected during the fruit development and ripening. These findings ultimately provide insights in the pathways which are essential during fruit development and ripening across species.
    Full-text · Article · Nov 2013 · Plant physiology
  • Source
    • "(available at JXB online). Ripe fruit were harvested from five independently transformed AroG 175 (two lines) and AroG 209 lines (three lines) and WT plants, and were analysed by an established high-resolution LC-MS-based metabolomics platform (negative ion-mode; Rogachev and Aharoni, 2012). PCA of whole ripe fruit extracts showed that the metabolic profile of the WT differed from those of the AroG 175 and AroG 209 lines (Fig. 2A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tomato (Solanum lycopersicum) fruit contains significant amounts of bioactive compounds, particularly multiple classes of specialized metabolites. Enhancing the synthesis and accumulation of these substances, specifically in fruits, are central for improving tomato fruit quality (e.g. flavour and aroma) and could aid in elucidate pathways of specialized metabolism. To promote the production of specialized metabolites in tomato fruit, this work expressed under a fruit ripening-specific promoter, E8, a bacterial AroG gene encoding a 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS), which is feedback-insensitive to phenylalanine inhibition. DAHPS, the first enzyme of the shikimate pathway, links between the primary and specialized metabolism derived from aromatic amino acids. AroG expression influenced the levels of number of primary metabolites, such as shikimic acid and aromatic amino acids, as well as multiple volatile and non-volatile phenylpropanoids specialized metabolites and carotenoids. An organoleptic test, performed by trained panellists, suggested that the ripe AroG-expressing tomato fruits had a preferred floral aroma compare with fruits of the wild-type line. These results imply that fruit-specific manipulation of the conversion of primary to specialized metabolism is an attractive approach for improving fruit aroma and flavour qualities as well as discovering novel fruit-specialized metabolites.
    Full-text · Article · Oct 2013 · Journal of Experimental Botany
  • Source
    • "Therefore, LC has the potential to analyse a wide variety of metabolites in plants. The recent development of ultra-performance liquid chromatography (UPLC) makes the technique more powerful because of its higher resolution, sensitivity and throughput than conventional high-performance liquid chromatography (HPLC) [16]. Electrospray ionisation (ESI) is widely used for ionisation to connect LC and MS. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Plant metabolism is perturbed by various abiotic stresses. As such the metabolic network of plants must be reconfigured under stress conditions in order to allow both the maintenance of metabolic homeostasis and the production of compounds that ameliorate the stress. The recent development and adoption of metabolomics and systems biology approaches enable us not only to gain a comprehensive overview, but also a detailed analysis of crucial components of the plant metabolic response to abiotic stresses. In this review we introduce the analytical methods used for plant metabolomics and describe their use in studies related to the metabolic response to water, temperature, light, nutrient limitation, ion and oxidative stresses. Both similarity and specificity of the metabolic responses against diverse abiotic stress are evaluated using data available in the literature. Classically discussed stress compounds such as proline, γ-amino butyrate and polyamines are reviewed, and the widespread importance of branched chain amino acid metabolism under stress condition is discussed. Finally, where possible, mechanistic insights into metabolic regulatory processes are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s00018-012-1091-5) contains supplementary material, which is available to authorized users.
    Preview · Article · Aug 2012 · Cellular and Molecular Life Sciences CMLS
Show more