Thylakoid phosphoproteins: identification of phosphorylation sites

Redox-dependent thylakoid protein phosphorylation regulates both the short- and long-term acclimation of the photosynthetic apparatus to changes in environmental conditions. The major thylakoid phosphoproteins belong to photosystem II (D1, D2, CP43, PsbH) and its light-harvesting antenna (Lhcb1, Lhcb2, CP29), but a number of minor phosphoproteins have also been identified. The detection methods traditionally include the radiolabeling techniques, electrophoretic separation of the phosphorylated and unphosphorylated forms of the protein, and the use of phosphoamino acid antibodies or phosphoprotein-specific dyes. The recent progress in mass spectrometry techniques and methods of proteomics allow for the successful identification and analyses of protein phosphorylation. In mass spectrometry approaches no exogenous tracer is needed and natural phosphorylation of proteins can be characterized with high sensitivity yielding the mapping of exact phosphorylation sites in the proteins as well. Various methods for the detection of thylakoid phosphoproteins, including the preparation of phosphopeptides for mass spectrometric analyses and techniques for phosphopeptide identification by electrospray ionization mass spectrometry (ESI-MS) are described. The experimental protocols for simultaneous identification of multiple phosphopeptides in complex peptide mixtures, enrichment of phosphopeptides by immobilized metal affinity chromatography (IMAC), and for their sequencing by tandem spectrometry are outlined.