Biomedical Engineering Reference
In-Depth Information
Time (min) %B
0
2
0.1
2
5
25
5.1
80
8
80
8.1
2
The composition of mobile phases A and B is given in
Section
2.4
. These settings allow analysing about 100 sam-
ples/day/mass spectrometer.
4. Run a series of standards together with the samples; this
will later be used to construct a standard curve in order to
perform an absolute quantification of the product. For this
purpose prepare serial dilutions of phospho-Aktide in 0.1%
formic acid: 0.05, 0.025, 0.1, 0.5, 5, 50, 125 (
μ
M) contain-
M of the IS peptide. Run these standards in parallel
with the unknown samples (
see
Note 26
).
5. Analyse 1
ing 0.1
μ
l of unknown samples and the standard serial
dilution samples by LC-MS/MS using the settings indicated
above.
6. Phospho-Aktide quantification in the different samples is
performed by measuring the area under the curve of the
extracted ion chromatogram for the fragment ion at
m
/
z
see
Note 27
). The obtained areas must be normalised by
the area of the phospho-Aktide internal standard fragment
at
m
/
z
403.7.
7. Results from the standards are used to generate a standard
curve by plotting phospho-Aktide/IS signal in the
y
-axis
and phospho-Aktide concentration in the
x
-axis. The result-
ing graph will allow to obtain a linear regression equation
(
y
μ
ax+b
, where
a
represents the slope of the curve,
b
is
the y-intercept,
y
is the normalised area at
m
/
z
400.7 and
x
is the micromolar concentration of phospho-Aktide) and
the correlation coefficient (
R
2
) value provides a measure of
the reliability of the linear relationship between the
x
and the
y
values (values close to 1 indicate excellent linear reliability).
8. By fitting the area values from unknown samples to the linear
Aktide generated by each individual reaction can be mea-
sured in absolute units. Finally, these results can be nor-
malised by the different protein content of the cell lysate
=
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