Selected Reaction Monitoring Applied to Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 164

single LC-MS run. The TSQ Vantage (Thermo Scientific)
has been used for the SRM experiments described in this
account.
2) The instrument needs to be properly calibrated and tuned
for peptide quantification. As quality control, a mixture of
peptide standards with well-defined concentrations (such as
1fmol/

L) is used to assess the HPLC separation and
instrument performances before analyzing biological sam-
ples.
3) The isotope-labeled peptides are also used to optimize the
experimental conditions to maximize the overall sensitiv-
ity, by fine-tuning specific instrument parameters such as
the collision energy and the source potentials. Synthetic
peptides in which stable isotopes are incorporated into the
C-terminal amino acid are commonly used as they have
become commercially available from several sources at rea-
sonable pricing. Alternative isotope dilution approaches are
emerging, including the addition of concatenated recom-
binant protein, referred to as QconCAT composed of the
peptides to be quantified ( 10 ) .
4) If a good correlation between MS/MS spectra measured
on an ion trap instrument and triple quadrupole instru-
ment has been demonstrated ( 11 ) , this allows the use of
library spectra from a different platform to design SRM
experiments.
5) For larger scale studies, i.e., analysis of large number of sam-
ples, the optimization of LC-MS will have a large impact
on the overall time. The complexity of the sample is often
the limiting factor. In essence, a relatively short gradient
would be preferred if the sample complexity accommodates
it. Most experiments in our hands were performed using
30- or 40-min gradients.

μ

Acknowledgments

Dr. Andreas Huhmer and David Fischer are gratefully acknowl-
edged for helpful discussion.

References

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