Biology Reference
In-Depth Information
of relative changes, it is sufficient to normalize the ratio to the amount of biomass
(
;
i
/
m
x
) assuming the cell morphology remained unchanged during the experiment.
In order to obtain absolute numbers for the intracellular concentrations, the
cytosolic volume of the biomass is required. In general, the intracellular metabolite
concentration as follows:
n
cell
i
V
cell
¼
n
i
V
Sample
OD
Sample
v
spec
where
V
Sample
is the total volume of extracted culture and OD
sample
is the OD
600
at
c
cell
i
¼
LL
1
OD
1
the time of sampling. The specific cell volume
v
spec
ð
in
m
Lmg
DryWeight
1
or
should be determined for the investigated strain and cultivation
conditions. Recently, it was shown that 1 mL sample of an
E. coli
culture with an
OD
600
of 1 has a total cell volume of 2.4-4.9
m
Þ
m
L depending on cultivation conditions
(
Volkmer and Heinemann, 2011
).
6
INTERPRETATION OF METABOLITE DATA
The possibilities for interpretation of metabolomics data verymuch depend on the level
of quantification that was achieved on the analytical side (
Table 5.1
). The metabolic
workflow described above is suitable for the generation of quantitative data on absolute
concentrations. If the calibration of themethod toexternal standards or the conversion to
intracellular concentrations based on cell volume is omitted, the generated data can per-
fectly well be used as relative-quantitative data. If U-
13
C-labelled internal standard is
omitted, the data can still be used as semi-quantitative data.
Table 5.1
Four Levels of Quantification in Metabolomics
Typical applications
Typical questions
Non-quantitative
Compound identification
Is metabolite A present in the sample?
Semi-quantitative
Very high-throughput
screening
Is the concentration of metabolite A in
sample X higher, lower or similar
compared to sample Y
Which metabolites exhibit the
greatest difference in concentration
between samples X and Y
Relative-
quantitative
Metabolomics as input to
statistical analyses
Does the concentration of metabolite
A correlate with environmental
parameter X? Can samples be
classified into groups?
Absolute-
quantitative
Integration with other
absolute data, kinetic
modelling,
thermodynamic
modelling
How does the concentration of
metabolite A relate to the
m
of
enzyme X? What thermodynamic
constraint does the concentration of
metabolite A impose on reaction Y?
K
