Basic Design of MRM Assays for Peptide Quantification - LC-MS/MS in Proteomics: Methods and Applications - page 176

in the initial phase. After the first rounds of analysis, additional
transitions can be added and low responding transitions can be
removed for subsequent rounds of optimization.
To show that the detected transitions originate from the
expected peptides, full MS/MS scans of the precursor ion can
be included ( 17 , 18 ) . The setup for such detection depends on
the specific instrument and software used. Subsequently, these
scans may also be manually inspected to ensure that the fragment
ions that were selected are the most abundant following CID.
Although it can be difficult to ensure optimal fragmentation for all
peptides in the MRM assay, inspecting the full MS/MS scans can
be used to optimize settings for fragmentation in the collision cell.
If a specific transition cannot readily be detected, the MRM
transition can be optimized with a synthetic peptide of the same
sequence. During later stages, an isotopically labeled version of
this peptide can be included to control for consistency between
the samples and for validation. Since AQUA- or SILAC-labeled
peptides have similar properties as the unlabeled peptide, the elu-
tion profile and fragmentation pattern will match the unlabeled
peptide. Thus, by selecting fragment ions from the y-ion series,
the ratio between the non-labeled and the isotopically labeled
peptide can be determined under the same experimental condi-
tions and used for quantification ( 14 ) .

3.3.Controls

After the initial MRM experiment has been set up and transitions
have been verified by full scans and/or by isotopically labeled pep-
tides, the MRM assay is ready for the next step. In this phase of
the experiment, it is important to incorporate controls for bio-
logical, analytical, and chromatographic variability. Addition of an
internal control at an early point during sample preparation can
be useful in order to track variability in sample preparation. For
example, adding a small amount of an extrinsic protein such as an
Escherichia coli protein to a human lysate prior to digestion can
be used to monitor digestion efficiency. Furthermore, adding a
small amount of control (for example, a peptide) just before sam-
ple analysis can be used to control for reproducibility of the LC
systems.
Injections of a blank as well as a control protein digest
between sample runs offer two additional controls. Injection of
a control sample following the analytical sample can be used
to ensure instrument calibration and chromatographic consis-
tency. Since the MRM assay only detects the peptides of inter-
est ( Fig. 10.3 ) , the effects from contaminants such as polymers
will be ignored; however, polymers can greatly affect the chro-
matographic consistency, and ionization efficiency, which is why
control injections are essential. Finally, a blank injection of buffer
A while running your specific MRM assay provides an important
control for chromatographic carry over.