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Metabolomic profiling of phytohormones in tomato: the crucial role of generic extraction and full-scan high resolution Orbitrap mass spectrometry

Lieven Van Meulebroek (UGent) , Kathy Steppe (UGent) and Lynn Vanhaecke (UGent)
Author
Organization
Project
IWT 101188
Abstract
Phytohormones are naturally occurring secondary metabolites and are of vital importance because of their regulating role in numerous plant processes such as germination, growth, reproduction, and protective response to stress. Since the plant’s developmental and physiological responses are resulting from considerable cross-talk and complex interaction of phytohormones, a metabolomic approach of the hormonal plant status is increasingly important.With the aim of metabolomic profiling the hormonal status of the tomato plant, an analytical method for the identification and quantification of phytohormones was developed. Such an approach, however, requires specific properties of both extraction procedure and mass detection method.The extraction procedure has to be kept as generic as possible, and should enable an efficient extraction of the targeted phytohormones without co-extraction of too much interfering substances. The final extraction procedure included an extraction phase with Bieleski solvent (methanol/water/formic acid, 75:20:5), a purification step using a 30 kDa Amicon centrifugal filter unit, and an enrichment phase by partial evaporation of the extraction solvent. The purification strategy was selected by evaluating various methods, including centrifugation, solid-phase extraction (Isolute C18 (EC), Sep-Pak and Bond Elut cartridges), syringe-driven filtration (0.45 μm Chromafil and 0.22 μm Millex units), and centrifugal filtration (10 and 30 kDa Amicon filter units).For mass analysis, the Orbitrap ExactiveTM mass spectrometer was selected because the high resolution (up to 100,000 FWHM) enables the identification and quantification of the low level phytohormones, present in a background of hundreds of more abundant compounds. In addition, the full scan principle of the Orbitrap mass spectrometer allows a metabolomic approach. The developed method was successfully validated accordingly Commission Directive EC/2002/657. Linearity (R2 > 0.99), repeatability Finally, the applicability of the analytical method for metabolomic profiling the hormonal plant status was demonstrated by using ToxID and Sieve software (Thermo Fisher Scientific), enabling an efficient screening of the full scan data.
Keywords
Validation, Orbitrap MS, Phytohormones, Metabolomics

Citation

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Chicago
Van Meulebroek, Lieven, Kathy Steppe, and Lynn Vanhaecke. 2012. “Metabolomic Profiling of Phytohormones in Tomato: The Crucial Role of Generic Extraction and Full-scan High Resolution Orbitrap Mass Spectrometry.” In Advances in Extraction Technologies, 14th International Symposium, Abstracts.
APA
Van Meulebroek, L., Steppe, K., & Vanhaecke, L. (2012). Metabolomic profiling of phytohormones in tomato: the crucial role of generic extraction and full-scan high resolution Orbitrap mass spectrometry. Advances in Extraction Technologies, 14th International symposium, Abstracts. Presented at the 14th International symposium on Advances in Extraction Technologies (ExTech 2012).
Vancouver
1.
Van Meulebroek L, Steppe K, Vanhaecke L. Metabolomic profiling of phytohormones in tomato: the crucial role of generic extraction and full-scan high resolution Orbitrap mass spectrometry. Advances in Extraction Technologies, 14th International symposium, Abstracts. 2012.
MLA
Van Meulebroek, Lieven, Kathy Steppe, and Lynn Vanhaecke. “Metabolomic Profiling of Phytohormones in Tomato: The Crucial Role of Generic Extraction and Full-scan High Resolution Orbitrap Mass Spectrometry.” Advances in Extraction Technologies, 14th International Symposium, Abstracts. 2012. Print.
@inproceedings{4237161,
  abstract     = {Phytohormones are naturally occurring secondary metabolites and are of vital importance because of their regulating role in numerous plant processes such as germination, growth, reproduction, and protective response to stress. Since the plant{\textquoteright}s developmental and physiological responses are resulting from considerable cross-talk and complex interaction of phytohormones, a metabolomic approach of the hormonal plant status is increasingly important.With the aim of metabolomic profiling the hormonal status of the tomato plant, an analytical method for the identification and quantification of phytohormones was developed. Such an approach, however, requires specific properties of both extraction procedure and mass detection method.The extraction procedure has to be kept as generic as possible, and should enable an efficient extraction of the targeted phytohormones without co-extraction of too much interfering substances. The final extraction procedure included an extraction phase with Bieleski solvent (methanol/water/formic acid, 75:20:5), a purification step using a 30 kDa Amicon centrifugal filter unit, and an enrichment phase by partial evaporation of the extraction solvent. The purification strategy was selected by evaluating various methods, including centrifugation, solid-phase extraction (Isolute C18 (EC), Sep-Pak and Bond Elut cartridges), syringe-driven filtration (0.45 \ensuremath{\mu}m Chromafil and 0.22 \ensuremath{\mu}m Millex units), and centrifugal filtration (10 and 30 kDa Amicon filter units).For mass analysis, the Orbitrap ExactiveTM mass spectrometer was selected because the high resolution (up to 100,000 FWHM) enables the identification and quantification of the low level phytohormones, present in a background of hundreds of more abundant compounds. In addition, the full scan principle of the Orbitrap mass spectrometer allows a metabolomic approach. The developed method was successfully validated accordingly Commission Directive EC/2002/657. Linearity (R2 {\textrangle} 0.99), repeatability 
Finally, the applicability of the analytical method for metabolomic profiling the hormonal plant status was demonstrated by using ToxID and Sieve software (Thermo Fisher Scientific), enabling an efficient screening of the full scan data.},
  author       = {Van Meulebroek, Lieven and Steppe, Kathy and Vanhaecke, Lynn},
  booktitle    = {Advances in Extraction Technologies, 14th International symposium, Abstracts},
  language     = {eng},
  location     = {Messina, Italy},
  title        = {Metabolomic profiling of phytohormones in tomato: the crucial role of generic extraction and full-scan high resolution Orbitrap mass spectrometry},
  year         = {2012},
}