Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli.
The global pool of all metabolites in a cell, or metabolome, is a reflection of all the metabolic functions of an organism under any particular growth condition. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. In the past, a variety of cell lysis methods were adopted for assays of individual metabolites or groups of intermediates in pathways. In this study, metabolites were extracted from Escherichia coli using six different commonly used procedures including acid or alkaline treatments, permeabilization by freezing with methanol, high-temperature extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol. Metabolites were extracted by the six methods from cells grown under identical conditions and labeled with [14C]glucose. The metabolomes were compared after 2-dimensional thin-layer chromatography of labeled compounds. For global analysis, extraction with cold (-40 degrees C) methanol showed the greatest promise, allowing simultaneous resolution of more than 95 metabolite spots. In contrast, 80 or less spots were obtained with other extraction methods. Extraction also influenced quantitative analysis of particular compounds. Metabolites such as adenosine exhibited up to 20-fold higher abundance after cold methanol extraction than after extraction with acid, alkali, or chloroform. The simplicity, rapidity, and universality of cold methanol extraction offer great promise if a single method of lysis is to be adopted in metabolome analysis.
Maharjan
Ram Prasad
RP
School of Molecular and Microbial Biosciences G08, University of Sydney, New South Wales, Sydney 2006, Australia.
Ferenci
Thomas
T
D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D000432:Methanol / Q000737:chemistry; D013696:Temperature
D002250:Carbon Radioisotopes; D000432:Methanol