UPLC-MS-based metabolite analysis in tomato.
ABSTRACT 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.
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ABSTRACT: Significant inter- and intraspecific genetic variation exists in duckweed, thus the potential for genome plasticity and manipulation is high. Polyploidy is recognised as a major mechanism of adaptation and speciation in plants. We produced several genome-duplicated lines of Landoltia punctata (Spirodela oligorrhiza) from both whole plants and regenerating explants using a colchicine-based cocktail. These lines stably maintained an enlarged frond and root morphology. DNA ploidy levels determined by florescence-activated cell sorting indicated genome duplication. Line A4 was analysed after 75 biomass doublings. Frond area, fresh and dry weights, rhizoid number and length were significantly increased versus wild type, while the growth rate was unchanged. This resulted in accumulation of biomass 17–20% faster in the A4 plants. We sought to determine if specific differences in gene products are found in the genome duplicated lines. Non-targeted ultra performance LC-quadrupole time of flight mass spectrometry was employed to compare some of the lines and the wild type to seek identification of up-regulated metabolites. We putatively identified differential metabolites in Line A65 as caffeoyl hexoses. The combination of directed genome duplication and metabolic profiling might offer a path for producing stable gene expression, leading to altered production of secondary metabolites.Plant Biology 07/2014; · 2.32 Impact Factor
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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.Journal of Experimental Botany 01/2013; · 5.79 Impact Factor
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ABSTRACT: Metabolite composition offers a powerful tool for understanding gene function and regulatory processes. However, metabolomics studies on multicellular organisms have thus far been performed primarily on whole organisms, organs, or cell lines, losing information about individual cell types within a tissue. With the goal of profiling metabolite content in different cell populations within an organ, we used FACS to dissect GFP-marked cells from Arabidopsis roots for metabolomics analysis. Here, we present the metabolic profiles obtained from five GFP-tagged lines representing core cell types in the root. Fifty metabolites were putatively identified, with the most prominent groups being glucosinolates, phenylpropanoids, and dipeptides, the latter of which is not yet explored in roots. The mRNA expression of enzymes or regulators in the corresponding biosynthetic pathways was compared with the relative metabolite abundance. Positive correlations suggest that the rate-limiting steps in biosynthesis of glucosinolates in the root are oxidative modifications of side chains. The current study presents a work flow for metabolomics analyses of cell-type populations.Proceedings of the National Academy of Sciences 03/2013; · 9.81 Impact Factor
UPLC-MS-based Metabolite Analysis in Tomato
Ilana Rogachev* and Asaph Aharoni
Department of Plant Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Recent advancements in the performance of hyphenated technologies based on ultra-pressure
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 (Ultra Performance Liquid Chromatography)-qTOF (quadruple time-of-flight) system
for profiling semi-polar metabolites in the model fruit plant tomato. Optimized extraction
method, instrument parameters and data treatment procedures are provided. The value of
UPLC instruments that 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 the system is most suitable, for extensive profiling of hundreds of plant metabolites.
Key Words: Tomato, fruit, UPLC, mass spectrometry; qTOF, metabolomics
*For correspondence: firstname.lastname@example.org
Tomatoes and tomato-based products are eaten throughout the world; their consumption is
believed to benefit the human health (1). Tomato metabolites, both primary and secondary, are
responsible for variations in fruit nutritional quality; therefore the analysis of tomato fruits
constituents is highly important. Another benefit from the analysis of the tomato metabolome
is that the metabolite data obtained can be interpreted in combination with new data arising
from the on-going tomato genome project (International Tomato Sequencing Project (2)),
which will lead to better understanding of gene functions.
Tomato fruit extracts contain carotenoids such as lycopene, β-carotene, and vitamin E, which
are known as effective antioxidants (3). Beside these lipophilic compounds, tomato tissues
comprise numerous semi-polar compounds: organic acids (mostly cinnamic acids), flavonoids
(mostly naringenin chalcone and glycosilated and acylated derivatives of quercetin and
kaempferol) and glycoalcaloids (tomatine, esculeosides) (4,5,6). HPLC and capillary
electrophoresis are the most widely used techniques for the separation of these classes of
compounds (7,8). UPLC (Ultra Performance Liquid Chromatography) instruments are based
on the use of small particle size chromatographic columns (less than 2 µm), offer substantial
resolution enhancement (9), and, hence, more effective separation of the compounds, reduction
of injection time and matrix effects. MS-based techniques, particularly in combination with
chromatographic technologies are most popular as these combine very high analytical precision
with equally high detection sensitivity (10). A TOF (time-of-flight) mass spectrometer, with
high resolution and mass accuracy, good dynamic range and a fast spectral acquisition capacity
(11), is most suitable in combination with UPLC, for extensive profiling of hundreds of plant
metabolites (12,13). In this chapter we will discuss the use of UPLC-qTOF for profiling semi-
polar metabolites in tomato tissues.
2.1 Reagents and equipment
1. Methanol, gradient grade (e.g. Merck, Cat. # 1.06007.2500).
2. Formic acid, spectroscopic grade (e.g. Fluka, Cat. # 06440).
3. Water, double deionized, from the Milli-Q purification system (Millipore, Bedford,
MA), resistivity 18.2 MΩ-cm, filtered through 0.2 µm membrane filter (see Note 1).
4. Acetonitrile, ultra gradient HPLC grade or LC-MS grade (e.g. JT Baker, Cat. # 9017).
5. Liquid nitrogen for grinding and freezing of tomato samples.
6. Standards for QC (quality control) samples: L-Tryptophan (Sigma, Cat. # T8941), L-
Phenylalanine (Sigma, Cat. # 78019), Chlorogenic acid (Fluka, Cat. # 25700), Caffeic
acid (Sigma, Cat. # C0625), p-Coumaric acid (Sigma, Cat. # C9008), Ferulic acid
(Aldrich, Cat. # 128708), Sinapic acid (Sigma, Cat. # D7927), Rutin hydrate (Sigma,
Cat. # R5143), Quercetin dihydrate (Sigma, Cat. # Q0125), Tomatine (Apin, Cat. #
03561t), Naringenin (Fluka, Cat. # 71155), Kaempferol (Fluka, Cat. # 60010).
7. IKA A11 basic grinder or a mortar and a pestle.
8. Screw-cap polypropylene (PP) tubes (50 ml) for storage of frozen samples (e.g.
Greiner, Greiner bio-one Inc.).
9. Screw-cap PP tubes (15 ml; e.g. Greiner) or 2-ml PP safe-lock eppendorf tubes for
10. Ultrasonic bath.
11. Single-use sterile latex-free syringes, 1 or 3-ml volume.
12. Single-use, 0.2 µm membrane syringe filters [e.g. diameter 4 mm PVDF (Millex-GV,
Cat # SLGVR04NL] or diameter 12 mm PTFE (PALL, Cat # 4552T) (see Note 2).
13. Amber-glass 2-ml autosampler vials and caps with a PTFE/Silicone septum. Use
suitable 250-ml glass inserts in case when you have a small volume of solution for
injection (less than 1 ml).
2.2 Instrumentation and software.
1. UPLC-PDA-qTOF system: UPLC Waters Acquity instrument connected in-line to an
Acquity PDA (photodiode array) detector and a Synapt HDMS detector (tandem
quadrupole/time-of-flight mass spectrometer). The MS detector is equipped with an
electrospray ion source (ESI). The Synapt HDMS system is operated in the standard
qTOF mode, without using the ion mobility capabilities (see Note 3).
2. UPLC BEH C18 column (Waters Acquity), 100 x 2.1 mm i.d., 1.7 µm, with a column
3. MassLynx 4.1 instrument software (Waters).
4. XCMS program (14) for mass peaks extraction and alignment (see Note 4).
2.3.1 For UPLC:
1. Mobile phase A: 5% acetonitrile/water (v/v), containing 0.1% formic acid (v/v).
2. Mobile phase B: 100% acetonitrile, containing 0.1% formic acid (v/v).
3. Strong needle wash solution: 80% methanol (a strong organic solution that
dissolves most components of the sample matrix).
4. Weak needle wash solution: 5% acetonitrile/water (v/v) (composition similar to the
initial conditions of the gradient).
5. Seal wash solution: 10% methanol/water (v/v).
2.3.2 Standard Mixture for a quality control sample (QC-Mix-12)
1. Prepare individual stock solutions of standard compounds (phenylalanine,
chlorogenic acid, caffeic acid, coumaric acid, ferulic acid, sinapic acid, rutin,
quercetin, naringenin and kaempferol) at a concentration of 1 mg/ml in methanol.
Prepare a tomatine stock solution at a concentration of 0.5 mg/ml in methanol.
Prepare tryptophan stock solution at a concentration of 1 mg/ml in 80% methanol
(v/v) containing 2% formic acid (v/v) (see Note 4). Sonicate stock solutions for
several minutes for better solubility of the compounds.
2. Prepare a stock mixture of standards by combining equal amounts of the individual
stock solutions. Each compound should be at the concentration of 83 µg/ml
(tomatine – 42 µg/ml). This solution can be stored at -20°C for 3 - 4 months without
significant changes in the concentration of the compounds.
3. Prepare the working standard mixture (QC-Mix-12) by diluting the stock mixture of
standards 10-fold with methanol. Final concentration of compounds is 8 µg/ml
(tomatine – 4 µg/ml). Use this solution as QC (quality control) and SST (system
suitability test) samples.
3.1 Sample preparation
The extraction of biological material with aqueous methanol has so far been the most widely
used option for LC-MS metabolite profiling schemes (15). Acidified aqueous methanol at a
final concentration of 75% methanol (v/v) and 0.1% formic acid (v/v) was considered to be the
most suitable solvent for efficient extraction of a wide range of secondary metabolites from
different plant species and tissues (16). A detailed description of the sample preparation
procedure can be found in (16). Tomato fruits contain relatively high concentrations of organic