Experimental analysis of receptor kinase phosphorylation.

Ligand binding by the extracellular domain of receptor kinases leads to phosphorylation and activation of the cytoplasmic domain of these important membrane-bound signaling proteins. To thoroughly characterize receptor kinase function, it is essential to identify specific phosphorylation sites by mass spectrometry. In this chapter, we summarize an efficient protein purification and modification protocol to prepare receptor kinases for liquid chromatography/tandem mass spectrometry analysis. Both recombinant receptor kinase cytoplasmic domains expressed in bacteria and full-length receptor kinase proteins expressed in living plant tissue are considered, and multiple methods of mass spectrometry are described that allow optimal identification of phosphorylated peptides of both in vitro- and in vivo-derived samples.