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to as intergel calibration. Both approaches are equally suited in SRM-calibrated 2DE,
as has been shown by
Maass
et al.
(2011)
(
Figure 3.4
).
Although the method of SRM-calibrated 2DE is already providing comprehen-
sive and generally precise data, there is still room for improvement. For example,
the current SRM-calibrated 2DE workflow could potentially be improved by using
difference gel electrophoresis (
¨
nl¨
et al.
, 1997
) to reduce the experimental error
introduced by the differential gel image analysis. Here, protein samples are stained
with different Cy-dyes that allow for the concurrent separation of up to three protein
samples on the same 2D gel. This would allow one of the fluorescence channels to be
used as a universal proteome reference for the determination of the absolute abun-
dance of the anchor proteins and the other two channels for the proteome samples
under investigation.
For systems biology approaches, any information regarding the absolute
abundance of proteins in a sample is interesting but not sufficient. The last step in
a systems biology proteomics approach will therefore consist of a combination of
information from the sample processing step and large-scale absolute quantification
step. This allows for the cell volume to be used to determine the amount of protein
yielded per cell and the application of correction factors that take into account losses
during cell disruption. Finally, the amount of each protein within a single cell, and
therefore the copy number per cell of each protein detected on the 2D gel, can be
calculated.
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