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Background  Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the  Arabidopsis  proteome, however, only a few studies look systematically for  in vivo  protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy.    Results  Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H 3 PO 4  neutral loss peaks and data-dependent MS 3 -fragmentation of the corresponding MS 2 -fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in  Arabidopsis thaliana  seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from  Chlamydomonas reinhardtii  cell cultures.    Conclusion  A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of  A. thaliana  and to proteins extracted from  C. reinhardtii . Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and  C. reinhardtii  in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.

plant methods

Combining metal oxide affinity chromatography (MOAC) and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites