Biology Reference
In-Depth Information
A complete unbiased approach for quantifi cation
independent from database search is mass accuracy precursor
alignment (MAPA) using the software ProtMax [
5
,
6
] (
http://
www.univie.ac.at/mosys/software.html
)
. A step-by-step tutorial
can be found at [
6
]. After enrichment techniques phosphopeptides
can be quantifi ed in a very convenient way in combination with
multivariate statistical analysis [
3
,
7
]. Multivariate statistics is of
paramount importance to distinguish constitutively phos-
phorylated peptides from phosphopeptides showing a clear
response to the treatment or regulatory effects [
3
,
7
]. We
routinely perform these analyses using COVAIN, a Matlab
®
-
based statistical toolbox that allows a deep statistical analysis and
data preprocessing in a user-friendly environment [
8
] (
http://
Further we recommend MaxQuant for phosphopeptide
identifi cation, phosphosite mapping, and quantifi cation of
phosphorylation stoichiometries. MaxQuant operation has
been described previously [
9
] and an excellent online user
forum is available via the software's homepage at
http://
maxquant.org/
. We describe some modifi cations to the default
settings for phosphopeptides.
Acknowledgments
We thank present and former colleagues from our labs, especially
Dennis Hopkins, Bastian Minkenberg, Matthias Nagler, Ella
Nukarinen, and Martin Thomas who contributed to the development
and optimization of the strategies described in this manuscript.
References
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‐
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