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Phosphoproteomics strategies for the functional analysis of signal transduction
Author(s) -
Morandell Sandra,
Stasyk Taras,
GrosstessnerHain Karin,
Roitinger Elisabeth,
Mechtler Karl,
Bonn Guenther K.,
Huber Lukas A.
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200600058
Subject(s) - phosphoproteomics , proteomics , phosphorylation , protein phosphorylation , affinity chromatography , signal transduction , computational biology , biology , kinase , biochemistry , protein kinase a , phosphoprotein , microbiology and biotechnology , enzyme , gene
Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low‐abundance kinase substrates, which can be achieved by various subcellular fractionation techniques. One strategy is the enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, e.g. immobilised metal affinity chromatography, prior to analysis. 2‐DE gels are powerful tools for the analysis of phosphoproteins when combined with new multiplexing techniques like DIGE, phosphospecific stains, autoradiography or immunoblotting. In addition, several gel‐free methods combining chromatography with highly sensitive MS have been successfully applied for the analysis of complex phosphoproteomes. Recently developed approaches like KESTREL or ’chemical genetics’ and also protein microarrays offer new possibilities for the identification of specific kinase targets. This review summarises various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates.