Selected Reaction Monitoring Applied to Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 162

4) Typically, for each peptide to be analyzed, three to five tran-
sitions are selected for an optimized SRM assay. In addition,
the elution time of each peptide is included in the method,
and the SRM analysis is performed in the t-SRM mode. The
time window for each peptide is user defined.

In high-sensitivity experiments, it is important to tune the instru-
ment parameters, in particular, the collision energy to maxi-
mize the signal of each transition. In our hands, the information
derived from an MS/MS spectrum is usually sufficient to estab-
lish an initial method to screen for the presence of peptides and
estimate their abundance. However, for the quantification of low-
abundance components, optimization of the collision energy is
often desirable to increase the signal-to-noise ratio.
1) To optimize the method, the experiment is repeated with
different parameters; the initial analysis is performed with
default collision energy setting (calculated using an empirical
formula: CE

3.7.Optimizationof
SRMExperiments

m / z value of the precursor).
2) In a second phase, the experiment is repeated with lower and
higher settings (e.g., 3 and 6 V below and above the default
value); the best conditions are retained for the final method.
The optimization often increases the sensitivity by a factor
of 2-3, which is not negligible for low-intensity signals ( see
Note 5 ).

= +2.0 × 0.034 ×

As mentioned earlier, typical proteomic experiments aim at mea-
suring a larger number of peptides in one single experiment.
Recent developments of instrument data acquisition software
enable us to use the observed or predicted elution time of a pep-
tide and to monitor the signals associated with that peptide only
during a small time window. This allows a more effective use of
the instrument time and will significantly increase the number of
transitions measured. The t-SRM method includes the instrument
parameters and the specific parameters associated with each tran-
sition.
1) Import the optimized SRM method, which includes infor-
mation of Q1 m / z ,Q3 m / z , collision energy, and retention
time for each transition.
2) Set the time window for the t-SRM experiment. The triple
quadrupole instrument is operated with a cycle time of 2 s,
the collision gas set at 1.2 mTorr, and a resolving power of
Q1 and Q3 of 0.7 unit (FWHM).
3) Set up the HPLC method, including the gradient.
4) Triplicate the t-SRM experiment using the optimized SRM
method.

3.8.Large-Scale
Experiment:
Time-BasedSRM
(T-SRM)