Biology Reference
In-Depth Information
Transfer 20 mL of the culture broth (OD
600
0.5) onto the middle of the
filter using a pipette. Try to load the filter evenly with cells and ensure that
the filter never runs dry during loading.
Wash the recovered cells with a fourfold diluted salt's medium containing
1gL
1
U-
13
C glucose—the diluted medium reduces the contamination of
the internal standard with salts.
Transfer the cell-loaded filters very quickly to the glass bottles with hot
ethanol using a pair of tweezers.
Incubate 3 min at 78
C.
Transfer the extract to a pre-cooled centrifuge tube on ice.
Separate cell debris and nitrocellulose from metabolite extract by
centrifugation at 12,000
4
C.
g
for 10 min at
20
C.
Repeat four times until the whole culture has been harvested (optional:
washing a fraction without carbon source will increase some low abundant
metabolites like cyclic AMP or phosphoenolpyruvate).
Vacuum dry the extract and re-suspend in 1 mL water.
Pool all cell extracts and remove proteins by ultra-filtration.
3. Spiking with U-
13
C-labelled standards and evaluation of results
Add 100
Store supernatant at
LofU-
13
C-labelled internal standard to the extraction solution.
Internal standard and sample are added at the same time to the extraction solution
(see
Section 4
).
Calculate the ratio
m
;
i
of the signal of unlabelled metabolite
i
and the signal of the
corresponding U-
13
C-labelled metabolite by mass spectrometry. This ratio equals
the moles of metabolite extracted from the cells
n
cell
i
ð
Þ
and moles of
U-
13
C-labelled metabolite from the internal standard
n
IS
i
ð
Þ
:
c
cel
i
V
cell
c
I
i
V
IS
where
V
cell
is the intracellular volume of the extracted biomass and
V
IS
is the volume
of internal standard added during extraction (here 100
n
cell
i
n
IS
i
¼
;
i
¼
m
L). In order to calculate the
intracellular metabolite concentration
c
cell
i
from this ratio, the concentration of the
c
IS
i
internal standard
is determined with a metabolite standard mixture, which is
prepared by dissolving the pure chemicals separately.
ð
Þ
4. Metabolite standard mixture
Prepare stock solutions of the pure metabolites separately, if possible, at a
concentration of 100 mM in water. For acids, neutralize the pH with ammonium.
To this end, weigh the pure compounds into a 1.5-mL Eppendorf-type tube using
an analytical balance. Use the calculated amount just as a reference and record the
exact amount. Then, calculate the final volume based on this amount and add
water or buffer accordingly.
Mix single compound standards such that every compound has a concentration
of 100
mol L
1
in the final volume. Add 10 mM ammonium acetate pH 7.2 and
m
