Selected Reaction Monitoring Applied to Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 163

3.9.Quantification
UsingStableIsotope
Dilution

1) The peptides used as internal standards are selected accord-
ing to the criteria discussed above, i.e., they are unique to
the targeted proteins, and present good mass spectrometric
properties. In addition, these peptides may fulfill additional
criteria regarding their composition, i.e., exclusion of amino
acids or sequences undergoing easy chemical modification,
i.e., oxidation of methionine residues or facile deamidation.
2) The 13 C stable isotopes are commonly incorporated into the
C-terminal arginine or lysine residues.
3) The data acquisition method is modified to include the tran-
sitions associated with the internal standards; this step can be
performed automatically by the method building software.
4) Add well-defined amounts of isotopically labeled peptide
(e.g., 1 fmol/ L) to the peptide digest to be analyzed.

5) The LC-MS analysis is performed in the t-SRM mode using
a method of targeting the endogenous and isotope-labeled
peptide pairs.
6) During the data analysis, the absolute amount of each tar-
geted peptide present in the sample is computed based on
the peak area ratio of the endogenous and isotope-labeled
peptide, and the amount of internal standard added to the
sample.
7) For rigorous quantification analysis, one might consider to
repeat the analysis with the internal standard at several con-
centrations in order to establish the linear response range.

μ

To evaluate the robustness of an optimized SRM method, repli-
cate analyses are performed.
1) The results from replicated runs (minimally triplicates) allow
determination of the precision of the method, both at the
individual transition level and at the peptide level.
2) Measurements that exhibit large variability should be
excluded; the ultimate assay will retain the transitions which
showed lowest coefficient of variance (CV).
3) Whenever possible, at least two peptides for each protein of
interest (covering different regions of the sequence) should
be analyzed to ensure reliability of the analyses.

3.10.Evaluating
theRobustness
of theMethod

4. Notes

1) Although, any triple quadrupole instrument is capable of
SRM measurements, a time-scheduling capability is essential
for monitoring a large number of SRM transitions during a