| Protein phosphorylation is a key cell signaling event crucial to the proper function and stabilization of the cell. Reversible phosphorylation helps mediate signals and protein-protein interactions, and identification of phosphorylation sites on proteins has helped elucidate cellular mechanisms. Mass spectrometric techniques are well-suited for the identification of phosphorylation sites on single proteins or on proteins from complex mixtures. Immobilized metal affinity chromatography (IMAC) is used to enrich low-level phosphopeptides from peptide mixtures, and the phosphopeptides are analyzed using electron transfer dissociation (ETD), a technique that lends itself to unambiguous phosphosite identification. Reversible protein phosphorylation is also a common event in the transport and of the plant hormone auxin, and with the goal of elucidating the mechanism of auxin transport in plants, the investigation of differential phosphorylation expression on proteins from Arabidopsis thaliana was undertaken using tools of mass spectrometry. Also, several phosphorylations sites have been identified on the human histone code mediator, Heterochromatin Protein 1 (HP1). HP1 is an evolutionarily conserved protein involved in epigenetic expression and has been implicated in the propagation of heterochromatin and gene silencing. The phosphopeptides were enriched using IMAC and fragmented by electron transfer dissociation (ETD) for phosphosite identification. |
