Biology Reference
In-Depth Information
Fig.
3
Dilution series for absolute quantifi cation of endogenous peptide levels.
Shown in fi lled symbols are two dilution series of externally calibrated stable
isotope labeled (
13
C
6
15
N
4
Arg) reference peptides NH
2
-ALYDNVAESPDELSFR- COOH
(fi lled circles) and NH
2
-FNSLNELVDYHR-COOH (fi lled triangles). The SRM-MS
measurements were carried out using a triple quadrupole (Thermo TSQ Vantage)
and each measurement, both the exogenous reference peptide and the endog-
enous peptide were measured. The concentration of endogenous peptides can
be inferred from the calibration curve. Together with the number of cells used to
obtain the tryptic peptides, absolute values can be determined
Another popular labeling method are stable isotope amino
acids
13
C
6
15
N
2
Lys or
13
C
6
15
N
4
Arg as reference standards for
tryptic protein digests. These stable isotope amino acids are
favorable, as H to D exchanges affect the C18 reverse phase
chromatography of the analytes [
42
]. On the protein level,
13
C
6
15
N
2
Lys or
13
C
6
15
N
4
Arg can be used as described in detail
by the Brun laboratory [
43
,
44
]. On the peptide level, crude
purifi ed
13
C
6
15
N
2
Lys or
13
C
6
15
N
4
Arg labeled peptides can be
added as reference standard to monitor changes of peptides,
and therefore proteins, as a function of perturbation. In case of
purifi ed externally calibrated (amino acid analysis) stable iso-
tope labeled reference peptides, absolute values can be obtained
if the same amount of endogenous lysate is measured in a dilu-
tion series of the reference peptide measuring the endoge-
nous/exogenous peptide pair (Fig.
3
) [
45
].
8. Concluding Remarks
Scheduled SRM-MS is a very powerful mass spectrometry
method allowing for reliable measurement of analytes [
35
].
Future developments will undoubtedly increase the dynamic
range of mass spectrometers capable of SRM-MS. A limitation
of SRM-MS is the limited number of analytes that can be
analyzed per LC-SRM-MS run. Using data-independent acqui-
sition of the peptidome, e.g., SWATH-MS [
36
], has the prom-
ise to monitor the peptidome which ionizes well in electrospray
ionization mass spectrometry. Also, the development of speedy
