Biology Reference
In-Depth Information
the flow equilibrium associated with the continuous supply and utilization of each
metabolite in the metabolic network.
In sharp contrast to other 'omics' methods that assess mRNA or protein abun-
dance, metabolomics provide a very fast and sensitive functional read-out that is
directly linked to bacterial physiology. The metabolome can respond within a second
or less to environmental changes (
Kresnowati
et al.
,2006
). This advantage comes,
however, with the major technical challenge that extracting intracellular metabolites
from bacteria is highly susceptible to errors and artefacts. Essentially, any change of
the physiochemical milieu, such as temperature, oxygen supply or pH, during the
sampling procedure can lead to measurable differences and can obscure the faithful
representation of the intracellular metabolite composition.
Here, we describe our current set of methods to obtain representative snapshots of
a specified set of metabolites in bacteria. Much more important than for other 'omics
methods', the analysis requires robust and standardized cultivation conditions that
maintain cellular homeostasis during sampling until metabolism is arrested. Our
protocol is structured into four steps of the workflow, for each of which we provide
several methods and comment on their pros and cons:
Cultivation
describes our standard protocols for (A) media, (B) inoculum and
(C) cultivation. The method is optimized for a fast filtration sampling to obtain
steady-state metabolite concentrations in bacteria.
Sampling
describes three methods of sampling for metabolome analysis: (A) fast
filtration for liquid shake flask cultures with low biomass concentration, (B)
whole cell broth extraction for sampling from cultures with high biomass
concentration and (C) high-throughput sampling from 96-well deep well plates.
Simultaneous extraction and inactivation of metabolism
describe two extraction
methods: (A) hot ethanol extraction for global metabolome analysis in one step
and (B) acidic acetonitrile extraction for quantification of nucleotide
triphosphates.
Analysis of metabolite extracts by a targeted mass spectrometry
describes our
analytical method consisting of (A) separation by liquid chromatography, (B)
detection by mass spectrometry and (C) peak integration. Further, we describe
how to obtain quantitative data by using (D) U-
13
C-labelled internal standards
and (E) an estimation of the cytosolic volume to calculate intracellular
concentrations.
In the last section, we briefly discuss how the absolute metabolite concentrations,
obtained as above, can be used to investigate the regulation of cellular
metabolism and other biological functions.
2
CULTIVATION OF BACTERIA FOR METABOLOME ANALYSIS
The metabolomics standard initiative recommends best practice reporting standards
to provide sufficient information about strains, inoculation procedure, cultivation
conditions and sampling time points to allow meaningful comparison of data sets
