Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry

The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13)...

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Bibliographic Details
Published in:	The Plant journal : for cell and molecular biology 2011-10, Vol.68 (2), p.364-376
Main Authors:	Giavalisco, Patrick, Li, Yan, Matthes, Annemarie, Eckhardt, Aenne, Hubberten, Hans-Michael, Hesse, Holger, Segu, Shruthi, Hummel, Jan, Köhl, Karin, Willmitzer, Lothar
Format:	Article
Language:	eng
Subjects:	Arabidopsis - chemistry Arabidopsis - metabolism Carbon Isotopes Chlorophyll - analogs & derivatives Chlorophyll - chemistry Databases, Factual Fourier Analysis Isotope Labeling Lipids - analysis Mass Spectrometry Metabolomics - methods Nitrogen Isotopes Plant Extracts - chemistry Plant Extracts - isolation & purification Plant Leaves - chemistry Plant Leaves - metabolism Plant Roots - chemistry Plant Roots - metabolism Proteomics - methods Reproducibility of Results Sulfur Isotopes
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Summary:	The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.
ISSN:	1365-313X