Biology Reference
In-Depth Information
and transfer the cultures to a low salt medium before sampling. This will reduce
contamination of the samples with salt and remove a substantial amount of the extra-
cellular metabolites that accumulate during cultivation. Such an approach, involving
a rapid exchange of cultivation medium, was recently applied on a larger scale for the
steady-state analysis of
E. coli
metabolism (
Link
et al.
, 2009
).
4
EXTRACTION OF METABOLITES FROM BACTERIA
The ideal extraction method should maximize the release of metabolites from the cell
and at the same time minimize the degradation or conversion of these compounds.
Extraction methods have involved combinations of freeze-thaw cycles, solvents,
boiling and extreme pH. Six different methods for extracting metabolites from
E. coli
were compared by
Maharjan and Ferenci (2003)
. They showed that the extrac-
tion methodology significantly influenced the results of metabolome analysis and, on
the basis of their findings, recommended a cold methanol extraction method for
global metabolome analysis. However, cold extraction does not always ensure
the efficient deactivation of enzymes as reflected in the decomposition of nucleotide
triphosphates when cold methanol/water was used (
Kimball and Rabinowitz, 2006
).
A recent study reported that boiling ethanol is a suitable one-step method for global
metabolome analysis, since the hot ethanol effectively extracts metabolites, dena-
tures and precipitates proteins, is easily removed by evaporation, and has a minimal
effect on pH (
Winder
et al.
, 2008
).
Here, we describe metabolite extraction involving hot ethanol/water (60:40) that
ensures broad coverage of metabolites, yet is fast (3 min) and convenient. However,
we also observed significant decomposition of nucleotide triphosphates in extracts
prepared with hot ethanol due to exchange of phosphate groups among nucleotide
phosphates. For this reason, we recommend acidic acetonitrile extraction to quantify
high-energy metabolites such as adenosine triphosphates (
Rabinowitz and Kimball,
2007
).
4.1
Hot ethanol extraction of filtered samples
Prepare 4 mL aliquots of 60:40 ethanol (p.a.)/water in small glass bottles and
preheat to 78
C in a water bath. Temperatures below the boiling point of ethanol
facilitate the liquid handling.
Transfer a nitrocellulose filter disc with sample into the extraction solution (see
Section 2
). Ensure the filter is completely immersed in the hot ethanol.
Add an internal standard (e.g. U-
13
C-labelled metabolite extract).
Incubate for 3 min at 78
C, shaking vigorously every minute.
Transfer the extract to a pre-cooled centrifuge tube on ice.
Separate cell debris and nitrocellulose from metabolite extract by centrifugation
at 12,000
g
for 10 min at
4
C.
