Biology Reference
In-Depth Information
spectrometry (MS) has been successfully applied to highly com-
plex crude proteomics samples [
1
,
2
]. In contrast to relative quan-
tification, absolute quantitative data result in absolute
concentration levels. Thus besides comparison of different experi-
mental treatments, absolute quantifi cation enables the analysis of
protein stoichiometry within a sample, the differentiation of iso-
forms as well as the comparison of inter-experimental conditions,
such as different species. Additionally, it leads to highly verifi able
data. In analogy to the well-known Western Blot, Lehmann et al.
[
3
] fi rst coined the suitable term Mass Western. While for Western
Blot analysis synthetic peptides can be used for the synthesis of
antibodies, they can be directly applied for the sensitive and tar-
geted detection and absolute quantifi cation using the Mass
Western. It is the integration of stable isotope labeled synthetic
peptides in combination with gel based or gel-free LC-SRM/MS.
A theoretical and an experimental approach to set up the Mass
Western can be distinguished (Fig.
1
). Both approaches start out
by defi ning at least one protein of interest. The theoretical
approach continues with in silico digestion of proteins and predic-
tion of proteotypic peptides (understood as a unique amino acid
sequence of a peptide, unambiguously identifying a specifi c pro-
tein of interest within a given proteome) for the target proteins
(in reference to the proteome of the sample). Subsequently, the
Choice of proteotypic peptides 3.2
Number of peptides per protein 3.3
Label position 3.4
Choice of transitions 3.5
Calibration curves 3.6
Absolute quantification 3.7
Frequently asked question:
Triple Quadrupole or Orbitrap 3.8
Fig.
1
Workfl ow diagram of the steps for Mass Western as described in the text
