Biology Reference
In-Depth Information
Mass Western
Western Blot
protein of interest
=> selection of proteotypic peptides
protein of interest
synthetic peptides/
protein overexpression
[
15
N/
13
C]synthetic peptides
immunisation/
polyclonals/monoclonals
tuning of mass spectrometer/
calibration curves/
absolute quantitation [pmol/fw]
immunserum/
purification of antibody
SDS-PAGE/2DE
SDS-PAGE/2DE
cut out molecular mass range/digest/
non-gel direct digestion
Blot
LC/MS
1. Antibody/2.Antibody/Staining
Fig.
3
MASS WESTERN strategy. Proteotypic peptides are initially selected from
a proteome analysis. Those peptides serve as model for synthetic internal stabile
isotope standards and are introduced for absolute quantifi cation using triple
quadrupole mass analyzer [
4
]. For comparison the typical strategy of a Western
blot using specifi c antibodies is shown
production of protein-specifi c antibodies it is very diffi cult to
distinguish between proteins of high homology leading to so-
called cross-reactivity. In addition with this technique absolute
quantifi cation of exact concentrations is almost impossible. We
developed a strategy based on mass spectrometry, which enables
to distinguish and to absolutely quantify protein isoforms or
whole pathways out of complex samples using stable isotope-
labeled synthetic peptides. Principles of the technique were fi rst
employed in the late 1980s [
15
]. Due to its similarity with the
Western Blot it is also called MASS WESTERN (Fig.
3
). The
MASS WESTERN allows for a high sample throughput and the
possibility to analyze many proteins (>100) within a single analy-
sis depending on how many proteotypic peptides per protein are
used. Proteome data measured in different cell states can be
integrated in a proteome database (e.g. ProMEX, Fig.
1
). They
comprise proteotypic peptide libraries as base for the MASS
WESTERN method design (Figs.
1
and
3
). It allows for the
detection of entire metabolic pathways such as Calvin Cycle,
Glycolysis, and citric acid cycle in parallel [
16
]. These data are
important for the mathematical reconstruction and prediction of
metabolic pathways and their regulatory mechanisms and hence
relevant for new insights in systems biology.
