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

Abstract

SUMMARYThe 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 highresolution 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.

Simultaneous extraction of metabolome and lipidome with methyl tert-butyl ether from a single small tissue sample for ultra-high performance liquid chromatography/mass spectrometry

“…Previously, only the polar fraction of MTBE extracts has been used for non-targeted metabolomic analyses, a strategy that has been described for plant material and plasma [28,29]. Aiming to apply non-targeted metabolomics to MTBE extracts of mammalian tissue and at the same time increasing the number of detectable metabolites, we extracted 10 mg of freeze-dried liver (6 replicates) and subjected aliquots of the mix and of the polar fraction to nontargeted metabolomic analyses.…”

Comprehensive Dissection of Spatiotemporal Metabolic Shifts in Primary, Secondary, and Lipid Metabolism during Developmental Senescence in Arabidopsis

“…In order to investigate metabolic changes, including the onset of metabolite remobilization, during developmental senescence in rosette leaves, upper leaves, and siliques, profiling of lipophilic metabolites by ultraperformance liquid chromatography (UPLC)-mass spectrometry (MS; Giavalisco et al, 2011) metabolites by HPLC and gas chromatography (GC)-MS (Lisec et al, 2006;Erban et al, 2007), and secondary metabolites by liquid chromatography (LC)-MS (Tohge and Fernie, 2010) was performed (Fig. 2).…”

Metabolomics applied in bioenergy

“…As for other analytic tools, the choice of methodology relies on the compound class of interest. For instance, GC-MS is extensively used for the analysis of semi-polar primary metabolites due to its heat stability, which is applicable for modifications through derivatization reactions that make them volatile [41], whereas LC-MS has been particularly employed for lipid or plant secondary metabolites [66][67][68]. In recent years, advances in high performance (e.g., fast GC and ultra-high performance liquid chromatography UHPLC) or multidimensional columns (e.g., GC × GC and LC × LC) have allowed for an increase in resolution, Applicable to large biomolecules with masses >200 kDa, sensitivity of several orders of magnitude providing faster analysis and better peak separation of complex biological mixtures [50].…”