Since protein phosphorylation was accepted to play a key role in cellular metabolism the investigation of this important posttranslational modification has gained ever-growing interest of researchers. However, until recently, technical challenges have made large scale studies on protein phosphorylation impossible. With dramatic improvements in both phosporylated peptides/proteins enrichment methods and in the selectivity and resolving power of mass spectrometry based techniques, the main challenges have been addressed and large scale protein phosphorylation studies are now being conducted. These technical advancements have also opened the door for the more accurate determination of actual protein phosphorylation sites. Consequently, another important aspect of this prominent signalling event can be investigated: proteins possess several phosphorylation sites and it is assumed that many of them have distinct regulatory function. While these processes are under thorough investigation in animals, protein phosphorylation in plants is still a developing field. This is particularly surprising in light of the enormous number of predicted protein kinases in the Arabidopsis thaliana genome exceeding apparently the complexity of animal systems. In this review, we illustrate the advantages and drawbacks of methods currently used for studying phosphorylation with an emphasis on phosphoprotein/peptide enrichment and mass spectrometry. We then describe how these methods can be used to reveal the biological importance of multisite protein phosphorylation in plants.
Keywords: Antibodies, phosphoserine, Immobilised Metal Affinity Chromatography, Chemical Derivatisation, Edman Sequencing, Imaging