Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized metal affinity chromatography, and TiO(2) were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized with a confidence level of 99% or higher. 41.4% of the identified proteins were allocated to the plasma membrane and about half of the phosphorylation sites have not been reported previously. A bioinformatic screen for 12 consensus sequences identified putative kinases for 642 phosphorylation sites. In summary, the protocol deployed here identified several hundred novel phosphorylation sites of cerebellar proteins. Furthermore, it provides a valuable tool to monitor the plasma membrane proteome from any small brain samples of interest under differing physiological or pathophysiological conditions.