Biology Reference
In-Depth Information
Add internal standard (e.g. U-
13
C-labelled metabolite extract).
20
C in a freezer or a thermo bath.
Incubate for 15 min at
Add 400
L of 15% NH
4
OH to neutralize the acidic extract.
Transfer the extract to a pre-cooled centrifuge tube.
Separate cell debris and nitrocellulose from the metabolite extract by
centrifugation at 12,000
m
4
C.
Dry the supernatants at 0.12 mbar to complete dryness in a vacuum concentrator.
Dry metabolite extracts can be stored at
g
for 10 min at
80
C until analysis.
Before analysis re-suspend the dry metabolite extracts in 100
m
L water centrifuge
4
C and use 10-20
at 12,000
g
for 10 min at
m
L of the supernatant for mass
spectrometry.
5
ANALYSIS BY MASS SPECTROMETRY
As metabolism generally takes place in an aqueous environment (e.g. cytosol), most
primary metabolites are hydrophilic and many posses ionic groups. Consequently,
chromatographic separation can take place in the gas phase after derivatization or
in liquid phase with normal phase, ion exchange, ion-pairing or hydrophilic interac-
tion chromatography (
Buescher
et al.
, 2009
). Note that metabolites containing
pyrophosphate groups (e.g. nucleotide triphosphates, NAD) can not be analyzed
by gas chromatography. Owing to the hydrophilic nature of most primary metabo-
lites, electron spray ionization (ESI) is the most widely used interface from liquid
chromatography to mass spectrometer. Of the various types of mass spectrometers
that are commercially available, ion traps and time-of-flight instruments are the most
versatile as they facilitate both quantification and compound identification by
accurate mass determination. For targeted analysis, triple quadrupole instruments
offer competitive sensitivity and a larger linear range of up to four orders of
magnitude.
Due to the large differences in abundance and physicochemical properties of
intracellular metabolites, no single published method can, to date, simultaneously
quantify the
1000 metabolites presumed to be present in a microbial cell. For
instance, in
E. coli
extracts, a combination of six methods was required for the quan-
tification of 176 metabolites (
van der Werf
et al.
, 2007a,b
) and a combination of two
methods quantified 103 metabolites (
Bennett
et al.
, 2009
). The ion-pairing LC triple
quadrupole MS method described here is particularly suited for the targeted analysis
of central carbon and energy metabolites (
Buescher
et al.
, 2010
). It covers a
wide range of metabolites (amino acids and precursors, sugar phosphates, organic
acids, nucleotides, redox cofactors, coenzyme A esters) while still allowing for
the separation of highly similar isomers such as sugar phosphates. Quantification
was demonstrated for 138 compounds in pure standards; 76-104 of which could also
be found in a diverse range of biological samples. As a prerequisite for this method,
metabolites of interest must be water soluble.
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