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IWT 101188
Abstract
Phytohormones are structurally and chemically diverse biomolecules, produced by plants. During a plant’s life cycle they fulfil a crucial role in various processes such as germination, growth, reproduction and protective response to biotic and abiotic stress. Phytohormones are classified into several classes including auxins, cytokinins, gibberellins, jasmonates, ethylene, abscisic acid, salicylic acid and brassinosteroids. Although to each class specific biological functions are assigned, there is mounting evidence of considerable cross-talk among plant-hormone-signalling pathways in regulating developmental and physiological processes. As a consequence interest has changed from the analysis of a specific phytohormone or a certain group of phytohormones to the multiple phytohormone analysis. The development of an analytical method, which allows quantification of all plant hormones in a given sample in a single experiment, would be highly desirable but is challenging however. After all, phytohormones are present at very low concentrations, in a background of a wide range of more abundant primary and secondary metabolites. Within this research the challenge was taken and it was tried to develop a method for the identification and quantification of all phytohormones in a tomato leaf or fruit. While keeping the metabolomic approach in mind, development of the extraction procedure was kept as generic as possible. Phytohormones from tomato tissue were first extracted with modified Bieleski solvent (methanol/ultrapure water/formic acid (70:20:5)), enriched with the antioxidant butylhydroxytoluene, followed by an inevitable purification step; in particular the usage of a microcon centrifugal filter device (30k). Depending on the considered extract, fruit or leaf, an evaporation phase for the enrichment of the target analytes was implemented. Chromatographic separation was performed with an U-HPLC system, equipped with an EC50/2 Nucleodur C18 Gravity column (1.8 μm, 50 x 2 mm) and with methanol and ultrapure water (0.1% formic acid) as mobile phases. Mass analysis was performed with the Fourier Transform Orbitrap Mass Spectrometer with characteristics most suitable for metabolomic profiling. After all, the high resolution (up to 100 000) of the Orbitrap makes it possible to deal with the relatively low level plant hormones and metabolites in the presence of hundreds of more abundant compounds, known to be present in plant extracts. In addition, although the method was developed by focussing on 8 phytohormones from 7 different classes (auxins, cytokinins, gibberellins, abscisic acid, salycilic acid, jasmonates and brassinosteroids) the full scan approach of the Orbitrap allows to screen in the plant extract for all known phytohormones and for unknown compounds with possible hormonal activity. The challenge to develop a suitable analysis method for the metabolomic profiling of phytohormones in (tomato) plants is almost completed. The final step consists out of the validation concerning the directive “Commission Decision 2002/657” of which the results will be presented. The possibilities of this innovative method are believed to be exceptional and most useful in the further elucidation of hormonal actions and hormone-induced signalling networks. Since phytohormones are of vital importance in divergent fields, the results of this research are undoubtedly widely applicable.
Keywords
Orbitrap MS, solid-liquid extraction, metabolomics, Phytohormones

Citation

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Chicago
Van Meulebroek, Lieven, Kathy Steppe, and Lynn Vanhaecke. 2012. “Metabolomic Profiling of Phytohormones in Tomato: Exploiting the High Resolution and Full Scan Characteristics of the Orbitrap Mass Spectrometer.” In 12th International Symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers (HTC-12) and 2nd International Symposium on Hyphenated Techniques for Sample Preparation (HTSP-2), Book of Abstracts, ed. Etienne Jooken. Koninklijke Vlaamse Chemische Vereniging (KVCV).
APA
Van Meulebroek, L., Steppe, K., & Vanhaecke, L. (2012). Metabolomic profiling of phytohormones in tomato: exploiting the high resolution and full scan characteristics of the Orbitrap mass spectrometer. In E. Jooken (Ed.), 12th International symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers (HTC-12) and 2nd International symposium on Hyphenated Techniques for Sample Preparation (HTSP-2), Book of abstracts. Presented at the 12th International symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers (HTC-12) ; 2nd International symposium on Hyphenated Techniques for Sample Preparation (HTSP-2), Koninklijke Vlaamse Chemische Vereniging (KVCV).
Vancouver
1.
Van Meulebroek L, Steppe K, Vanhaecke L. Metabolomic profiling of phytohormones in tomato: exploiting the high resolution and full scan characteristics of the Orbitrap mass spectrometer. In: Jooken E, editor. 12th International symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers (HTC-12) and 2nd International symposium on Hyphenated Techniques for Sample Preparation (HTSP-2), Book of abstracts. Koninklijke Vlaamse Chemische Vereniging (KVCV); 2012.
MLA
Van Meulebroek, Lieven, Kathy Steppe, and Lynn Vanhaecke. “Metabolomic Profiling of Phytohormones in Tomato: Exploiting the High Resolution and Full Scan Characteristics of the Orbitrap Mass Spectrometer.” 12th International Symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers (HTC-12) and 2nd International Symposium on Hyphenated Techniques for Sample Preparation (HTSP-2), Book of Abstracts. Ed. Etienne Jooken. Koninklijke Vlaamse Chemische Vereniging (KVCV), 2012. Print.
@inproceedings{4251667,
  abstract     = {Phytohormones are structurally and chemically diverse biomolecules, produced by plants. During a plant{\textquoteright}s life cycle they fulfil a crucial role in various processes such as germination, growth, reproduction and protective response to biotic and abiotic stress. Phytohormones are classified into several classes including auxins, cytokinins, gibberellins, jasmonates, ethylene, abscisic acid, salicylic acid and brassinosteroids. Although to each class specific biological functions are assigned, there is mounting evidence of considerable cross-talk among plant-hormone-signalling pathways in regulating developmental and physiological processes. As a consequence interest has changed from the analysis of a specific phytohormone or a certain group of phytohormones to the multiple phytohormone analysis. The development of an analytical method, which allows quantification of all plant hormones in a given sample in a single experiment, would be highly desirable but is challenging however. After all, phytohormones are present at very low concentrations, in a background of a wide range of more abundant primary and secondary metabolites.
Within this research the challenge was taken and it was tried to develop a method for the identification and quantification of all phytohormones in a tomato leaf or fruit. While keeping the metabolomic approach in mind, development of the extraction procedure was kept as generic as possible. Phytohormones from tomato tissue were first extracted with modified Bieleski solvent (methanol/ultrapure water/formic acid (70:20:5)), enriched with the antioxidant butylhydroxytoluene, followed by an inevitable purification step; in particular the usage of a microcon centrifugal filter device (30k). Depending on the considered extract, fruit or leaf, an evaporation phase for the enrichment of the target analytes was implemented. Chromatographic separation was performed with an U-HPLC system, equipped with an EC50/2 Nucleodur C18 Gravity column (1.8 \ensuremath{\mu}m, 50 x 2 mm) and with methanol and ultrapure water (0.1\% formic acid) as mobile phases. Mass analysis was performed with the Fourier Transform Orbitrap Mass Spectrometer with characteristics most suitable for metabolomic profiling. After all, the high resolution (up to 100 000) of the Orbitrap makes it possible to deal with the relatively low level plant hormones and metabolites in the presence of hundreds of more abundant compounds, known to be present in plant extracts. In addition, although the method was developed by focussing on 8 phytohormones from 7 different classes (auxins, cytokinins, gibberellins, abscisic acid, salycilic acid, jasmonates and brassinosteroids) the full scan approach of the Orbitrap allows to screen in the plant extract for all known phytohormones and for unknown compounds with possible hormonal activity. The challenge to develop a suitable analysis method for the metabolomic profiling of phytohormones in (tomato) plants is almost completed. The final step consists out of the validation concerning the directive {\textquotedblleft}Commission Decision 2002/657{\textquotedblright} of which the results will be presented. The possibilities of this innovative method are believed to be exceptional and most useful in the further elucidation of hormonal actions and hormone-induced signalling networks. Since phytohormones are of vital importance in divergent fields, the results of this research are undoubtedly widely applicable.},
  author       = {Van Meulebroek, Lieven and Steppe, Kathy and Vanhaecke, Lynn},
  booktitle    = {12th International symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers (HTC-12) and 2nd International symposium on Hyphenated Techniques for Sample Preparation (HTSP-2), Book of abstracts},
  editor       = {Jooken, Etienne},
  isbn         = {9789081124843},
  language     = {eng},
  location     = {Brugge, Belgium},
  publisher    = {Koninklijke Vlaamse Chemische Vereniging (KVCV)},
  title        = {Metabolomic profiling of phytohormones in tomato: exploiting the high resolution and full scan characteristics of the Orbitrap mass spectrometer},
  year         = {2012},
}