Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co‐regulation in biochemical networks

Abstract

A novel extraction protocol is described with which metabolites, proteins and RNA are sequentially extracted from the same sample, thereby providing a convenient procedure for the analysis of replicates as well as exploiting the inherent biological variation of independent samples for multivariate data analysis. A detection of 652 metabolites, 297 proteins and clear RNA bands in a single Arabidopsis thaliana leaf sample was validated by analysis with gas chromatography coupled to a time of flight mass spectrometer for metabolites, two-dimensional liquid chromatography coupled to mass spectrometry for proteins, and Northern blot analysis for RNA. A subset of the most abundant proteins and metabolites from replicate analysis of different Arabidopsis accessions was merged to form an integrative dataset allowing both classification of different genotypes and the unbiased analysis of the hierarchical organization of proteins and metabolites within a real biochemical network.

SWAPDT: A method for Short-time Withering Assessment of Probability for Drought Tolerance in Camellia sinensis validated by targeted metabolomics

“…accumulation of osmolytes in a response to drought stress (Slama et al, 2015). Hyperosmotic stress produces osmolytes which include polyols and sugars (mannitol, sorbitol and trehalose) and amino acids (proline and betaine) (Weckwerth et al, 2004). These compounds are water soluble and non-toxic at high concentrations.…”

An optimized procedure for metabonomic analysis of rat liver tissue using gas chromatography/time-of-flight mass spectrometry

“…Tissue-targeted metabonomic analyses have been carried out on brain tissue [11][12][13], liver tissue [14][15][16], and kidney tissue [17][18][19]. Metabolite extraction is a critical step in tissue metabonomic study, and the chloroform-methanol-water solvent system is considered the preferred method in several reports [20][21][22][23]. The latest relevant study was conducted by Wu et al [24], in which fish liver was used to optimize the extraction strategy of the preferred solvent system for NMR-and FT-ICR MS-based metabonomic studies.…”

Dynamic Adaption of Metabolic Pathways during Germination and Growth of Lily Pollen Tubes after Inhibition of the Electron Transport Chain

“…Preparation of extracts followed the method developed by Weckwerth et al (2004), with slight modifications. Pollen grains (60 mg) were incubated in Med M for 0, 10, 30, 60, 75, 90, 120, and 240 min (samples 0, 10, 30, 60, 75, 90, 120, and 240).…”