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.
- SourceAvailable from: Sofia Moco[show abstract] [hide abstract]
ABSTRACT: For the description of the metabolome of an organism, the development of common metabolite databases is of utmost importance. Here we present the Metabolome Tomato Database (MoTo DB), a metabolite database dedicated to liquid chromatography-mass spectrometry (LC-MS)- based metabolomics of tomato fruit (Solanum lycopersicum). A reproducible analytical approach consisting of reversed-phase LC coupled to quadrupole time-of-flight MS and photodiode array detection (PDA) was developed for large-scale detection and identification of mainly semipolar metabolites in plants and for the incorporation of the tomato fruit metabolite data into the MoTo DB. Chromatograms were processed using software tools for mass signal extraction and alignment, and intensity-dependent accurate mass calculation. The detected masses were assigned by matching their accurate mass signals with tomato compounds reported in literature and complemented, as much as possible, by PDA and MS/MS information, as well as by using reference compounds. Several novel compounds not previously reported for tomato fruit were identified in this manner and added to the database. The MoTo DB is available at http://appliedbioinformatics.wur.nl and contains all information so far assembled using this LC-PDA-quadrupole time-of-flight MS platform, including retention times, calculated accurate masses, PDA spectra, MS/MS fragments, and literature references. Unbiased metabolic profiling and comparison of peel and flesh tissues from tomato fruits validated the applicability of the MoTo DB, revealing that all flavonoids and alpha-tomatine were specifically present in the peel, while several other alkaloids and some particular phenylpropanoids were mainly present in the flesh tissue.Plant physiology 09/2006; 141(4):1205-18. · 6.56 Impact Factor
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ABSTRACT: Metabolite profiling in biomarker discovery, enzyme substrate assignment, drug activity/specificity determination, and basic metabolic research requires new data preprocessing approaches to correlate specific metabolites to their biological origin. Here we introduce an LC/MS-based data analysis approach, XCMS, which incorporates novel nonlinear retention time alignment, matched filtration, peak detection, and peak matching. Without using internal standards, the method dynamically identifies hundreds of endogenous metabolites for use as standards, calculating a nonlinear retention time correction profile for each sample. Following retention time correction, the relative metabolite ion intensities are directly compared to identify changes in specific endogenous metabolites, such as potential biomarkers. The software is demonstrated using data sets from a previously reported enzyme knockout study and a large-scale study of plasma samples. XCMS is freely available under an open-source license at http://metlin.scripps.edu/download/.Analytical Chemistry 03/2006; 78(3). · 5.70 Impact Factor
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ABSTRACT: The cuticle, covering the surface of all primary plant organs, plays important roles in plant development and protection against the biotic and abiotic environment. In contrast to vegetative organs, very little molecular information has been obtained regarding the surfaces of reproductive organs such as fleshy fruit. To broaden our knowledge related to fruit surface, comparative transcriptome and metabolome analyses were carried out on peel and flesh tissues during tomato (Solanum lycopersicum) fruit development. Out of 574 peel-associated transcripts, 17% were classified as putatively belonging to metabolic pathways generating cuticular components, such as wax, cutin, and phenylpropanoids. Orthologs of the Arabidopsis (Arabidopsis thaliana) SHINE2 and MIXTA-LIKE regulatory factors, activating cutin and wax biosynthesis and fruit epidermal cell differentiation, respectively, were also predominantly expressed in the peel. Ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer and gas chromatography-mass spectrometry using a flame ionization detector identified 100 metabolites that are enriched in the peel tissue during development. These included flavonoids, glycoalkaloids, and amyrin-type pentacyclic triterpenoids as well as polar metabolites associated with cuticle and cell wall metabolism and protection against photooxidative stress. Combined results at both transcript and metabolite levels revealed that the formation of cuticular lipids precedes phenylpropanoid and flavonoid biosynthesis. Expression patterns of reporter genes driven by the upstream region of the wax-associated SlCER6 gene indicated progressive activity of this wax biosynthetic gene in both fruit exocarp and endocarp. Peel-associated genes identified in our study, together with comparative analysis of genes enriched in surface tissues of various other plant species, establish a springboard for future investigations of plant surface biology.Plant physiology 07/2008; 147(2):823-51. · 6.56 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: email@example.com
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