Analysis of Protein Phosphorylation in Halobacterium salinarum

Protein phosphorylation plays an important role in the two-component system based signal transduction pathways in bacteria and in archaea. These cascades have been identified also in the extremely halophilic archaeon Halobacterium salinarum and are well understood. Beside these protein phosphorylation events in which phosphorylation of histidine and aspartate residues are crucial, only little is known about further protein phosphorylation in Halobacterium salinarum. From genome and proteome analysis on H. salinarum several putative protein kinases and phosphatases have been annotated and identified. Nevertheless, the extent of regulatory protein phosphorylation is not yet well elucidated. In order to identify phosphorylated proteins and to determine their phosphorylation sites different specific experimental approaches have been applied to the whole cell extract. A gel based approach utilizing 1and 2-dimensional gel electrophoresis after radioactive labeling of the cells with 32P resulted the identification of a number of phosphate carrying proteins by MALDI-TOF MS. In addition, anti-phosphoserine/threonine antibodies, were used for the determination of the phosphorylated proteins in both procedures (1and 2-DE). Discrete signals were observed indicating Ser/Thr phosphorylation but the magnitude of protein phosphorylation appeared to be rather low. Alternatively, a gel-free method involve strong cation exchange (SCX) chromatography was applied to enrich phosphopeptides resulted after trypsin digestion of the whole cell extract, under low salt elution conditions. LC-MS/MS analysis of the additional IMAC-enriched SCX fractions yielded the identification of threonine phosphorylation sites of different halobacterial proteins, the first archaeal Ser/Thr phosphorylation sites ever described. The question if the identified phosphorylation sites are of regulatory importance or just present metabolic intermediates has to be answered.