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compared to the spiked-in heavy-labelled peptide, calibration curves have to be
acquired using the optimized parameters in order to determine the limits of detection
and quantification (
Armbruster
et al.
, 1994; Zorn
et al.
, 1997
). Once the calibration
curves are established, they can be used to determine the peptide-specific linear
range of the transition signal versus the peptide concentration of a sample. Addition-
ally, heavy-labelled peptides should be introduced to the protein sample at levels
close to the expected natural levels to ensure that the sample concentration falls
within range of the assays linear response (
Gallien
et al.
, 2011
). Whereas this is eas-
ily achieved for AQUA, a trade-off is necessary for the QconCAT-derived peptides
that are inherently equimolar and therefore their concentration should be adjusted so
as to average out over the expected concentration range of the sample peptides. This
drawback can be limited if the expected protein concentrations are known prior to the
beginning of the analyses, because then proteins of a similar concentration range can
be grouped into the same QconCAT protein.
For the quantitative work, a known amount of the heavy peptides or QconCAT is
spiked into the proteome sample prior to proteolytic digestion. It is important that a
defined amount of protein extract is used if the same sample has previously been
subjected to differential gel image analysis. For the targeted measurement of the light
and heavy peptides with the same optimized SRM assays, the areas under the curves
for the two counterparts are used for the determination of the absolute expression
level of the peptide and therefore of the concentration of the protein in the sample
(
Kirkpatrick
et al.
, 2005; Gallien
et al.
, 2011; Kettenbach
et al.
, 2011
).
The overall goal of setting up the targeted analysis is to determine the best transition
pairs, with respect to the optimized instrument parameters and specificity in the chosen
retention time window, over other ion species that may be found also in a targeted SRM
approach. The quantitative data derived from experiments based on AQUA peptides or
on the QconCAT approach give information about the concentration of a given peptide
in the interrogated sample, which can also serve as the basis for calculating the absolute
abundance of related proteins in the proteome sample (see below).
5
GENERATION OF LARGE-SCALE RELATIVE PROTEOMICS
DATA: DIFFERENTIAL 2D GEL IMAGE ANALYSIS
Differential 2D gel image analysis is a widely used method for generating large-scale
relative quantitative proteomics abundance data. Despite being the oldest proteomics
method, 2DE is still a workhorse for laboratories that rely on its distinct advantages
(
Rabilloud
et al.
, 2010
).
2DE of proteins is technically characterized by a continuous separation space
within predefined
p
I- and MW ranges, performed by an electrophoretic step during
isoelectric focusing (IEF) and subsequent resolution by MW by gel electrophoresis
(
Carrette
et al.
, 2006
). The main advantages of 2DE, compared to other protein
separation approaches, are the direct accessibility and visualization of protein modi-
fications without the need to predict them, the reasonable financial and time costs,
