Biology Reference
In-Depth Information
The traces of some target metabolites can contain additional peaks of unknown
metabolites. As retention times are stable within a few seconds for consecutive
injections of samples of similar composition, the correct peak can easily be
identified by comparing the retention time to that in a chemically pure standard.
5.4
Isotope ratio-based quantification of intracellular metabolite
concentrations
Several isotope ratio-based approaches have been proposed for metabolome anal-
ysis (
Mashego
et al.
, 2004; Bennett
et al.
, 2008
). The general principle is that the
amount of isotopically labelled internal standard and analyte is quantified sepa-
rately using MS-based metabolomics and the ratio of both measurements is used
for all further calculations. The internal standard is spiked into the solution of the
extract right after sampling and undergoes similar degradation and loss during
sample preparation. Isotopologues are ideal internal standards in MS-based meta-
bolomics since standard and analyte have the same physicochemical properties but
different masses. U-
13
C isotopologues of all metabolites are relatively easily
obtained from cells grown on minimal medium with U-
13
C glucose as the sole
carbon source. Usually, U-
13
C-labelled cell extracts are obtained from yeast cul-
tivated in a fed-batch fermentation on 100% U-
13
C-labelled substrates (
Wu
et al.
,
2005
). Here, we first describe a yeast fermentation to obtain large quantities of
internal standard within 3 days of work. The second method describes a quick
anaerobic cultivation to obtain labelled cell extract within 1 day of work.
1. Batch fermentation of yeast on U-
13
C-labelled glucose
This method describes fermentation in a bioreactor to obtain 1 L of
U-
13
C-labelled yeast culture.
U-
13
C glucose minimal medium
The minimal medium used for the fed-batch cultivation of yeast is composed
per litre of
200 mL of a heat-sterilized base salt solution containing: 25 g L
1
(NH
4
)
2
SO
4
, 2.5 g L
1
MgSO
4
7H
2
O and 15.0 g L
1
KH
2
PO
4
.
751 mL filter-sterilized KH-phthalate-water (10 mM, pH 5).
1 mL of heat-sterilized vitamin solution containing 50 mg L
1
biotin,
1gL
1
Ca-pantothenate, 1 g L
1
nicotinic acid, 25 g L
1
inositol, 1 g L
1
pyridoxine, 200 mg L
1
p
-aminobenzoic acid, 1 g L
1
thiamine.
10 mL of a filter-sterilized trace element solution containing: 0.15 g L
1
EDTA,0.45gL
1
ZnSO
4
7H
2
O, 0.03 g L
1
CoCl
2
6H
2
O, 0.10 g L
1
4H
2
O, 0.03 g L
1
CuSO
4
2H
2
O, 0.30 g L
1
MnCl
2
5H
2
O, 0.45 g CaCl
2
7H
2
O, 0.04 g L
1
NaMoO
4
2H
2
O, 0.10 g L
1
H
3
BO
3
,0.01gL
1
KI.
FeSO
4
40 mL of filter-sterilized U-
13
C glucose solution (250 g L
1
).
Bioreactor set-up
2.1 L bioreactor with two Rushton turbines (e.g. Bioengineering KLF).
