Stable Isotope-Assisted Evaluation of Different Extraction Solvents for Untargeted Metabolomics of Plants

Abstract

The evaluation of extraction protocols for untargeted metabolomics approaches is still difficult. We have applied a novel stable isotope-assisted workflow for untargeted LC-HRMS-based plant metabolomics , which allows for the first time every detected feature to be considered for method evaluation. The efficiency and complementarity of commonly used extraction solvents, namely 1 + 3 (v/v) mixtures of water and selected organic solvents (methanol, acetonitrile or methanol/acetonitrile 1 + 1 (v/v)), with and without the addition of 0.1% (v/v) formic acid were compared. Four different wheat organs were sampled, extracted and analysed by LC-HRMS. Data evaluation was performed with the in-house-developed MetExtract II software and R. With all tested solvents a total of 871 metabolites were extracted in ear, 785 in stem, 733 in leaf and 517 in root samples, respectively. Between 48% (stem) and 57% (ear) of the metabolites detected in a particular organ were found with all extraction mixtures, and 127 of 996 metabolites were consistently shared between all extraction agent/organ combinations. In aqueous methanol, acidification with formic acid led to pronounced pH dependency regarding the precision of metabolite abundance and the number of detectable metabolites, whereas extracts of acetonitrile-containing mixtures were less affected. Moreover, methanol and acetonitrile have been found to be complementary with respect to extraction efficiency. Interestingly, the beneficial properties of both solvents can be combined by the use of a water-methanol-acetonitrile mixture for global metabolite extraction instead of aqueous methanol or aqueous acetonitrile alone.

Metabolomics in the Context of Plant Natural Products Research: From Sample Preparation to Metabolite Analysis

“…As a cleaner and safer solvent, methyl tert-butyl ether (MTBE) has also been used as an alternative to chloroform for liquid-liquid extraction [50], particularly for the recovery of metabolites and lipids from bacterial, plant, algal, flies, and diverse mammalian samples [41,42,[51][52][53][54][55][56][57][58][59][60]. The use of isotope-labeled internal standards during sample preparation is recommended to assess matrix effects [39,61]. For the selection of suitable solvent(s) and an extraction method that is suitable for different metabolite classes, readers are referred to the available extraction protocol reviews [32,35,39,40,62,63].…”

Preparation of uniformly labelled 13C- and 15N-plants using customised growth chambers

“…One is used for the determination of the The wheat ears were milled to a fine powder with a ball mill (MM400, Retsch, Haan, Germany) while being kept in frozen condition. The extraction was performed similarly as reported in (8…”

“…application are nutrition (1), evaluation of biosynthetic pathways (2), metabolic engineering (3), flux analysis (2,4), accurate quantification of plant metabolites (5), studies on plant-microbe interactions (6) and untargeted metabolomics and proteomics (6)(7)(8)(9)(10)(11)(12). The increasing popularity of isotopically labelled plant material goes hand in hand with the development of complementary analytical tools e.g.…”

“…Determined degree of enrichment after cultivation of 13 C-and 15 N-labelled T. aestivum and T. durum cultivars.Extracts of the stable isotopically labelled wheat ( 13 C and 15 N separately) were used to internally standardise native wheat extracts. These sample mixtures were measured with LC-HRMS and processing of the obtained raw data was performed with the in-house developed MetExtract II software(7,8,14,34). The program automatically performs an efficient filtering of all non-wheat derived metabolites by extracting metabolic features based on their chromatographic coelution and isotopic pattern (which is characteristic to the mixture of native and labelled extracts)(14).…”

Preparation of uniformly labelled 13C- and 15N-plants using customised growth chambers

“…The use of plants and plant-derived metabolites labelled with heavy stable isotopes offers an interesting perspective in phytochemical research. Some research fields where stable isotopes found application are nutrition (1), evaluation of biosynthetic pathways (2), metabolic engineering (3), flux analysis (2,4), accurate quantification of plant metabolites (5), studies on plant-microbe interactions (6) and untargeted metabolomics and proteomics (6)(7)(8)(9)(10)(11)(12). The increasing popularity of isotopically labelled plant material goes hand in hand with the development of complementary analytical tools e.g.…”